Home

Hotlink to MACSQuant User Manual

1. 4 Coiteate uptake unit Calibrate rack detection Show backup registry Update software Remove external media Time and date MACSQuant live support Import E xport user settings Uptake unit calibration 1 First push the Start calibration button to begin the calibration be careful the hardware moves 2 Move the needle and sampler by the hand to the specified position and press Save calibration 3 Test your calibration using the Test button select position you want to test __ strtcaibraton __ Seve calibration Start calibration Save calibration Zattet 0 S __ Startcalibration __ Save calibration _ f bU I Stat caibreton _ _Sevecatbraion C ten Soncaibraion J __Savecaitraion J Tet Figure 3 17 Calibration of the uptake unit is underway The yellow closed circle indicates that the needle arm may be freely moved for manual calibration 3 Manually adjust the y axis of the needle arm by moving it towards you taking care to ensure that the needle is correctly positioned over the center of the sheath flow port 4 Gently lower the needle arm into the sheath flow port until the needle makes first contact with the bottom of the orifice The needle should always remain perpendicular to the horizontal plane i e should not be arched or inserted diagonal ly Sheath flow port Figure 3 18 Inserting the
2. Figure 6 78 Three files were opened for grouping 175 2 Using the Samples menu _ highlight each file that must be grouped 3 Right click and select Group Samples Experiment Tools Channels Sample Statistic Cells live 0 0 0 adm20039 09 01_2022 003 100 0 3035 adm2003 03 01_2022 002 0 0 0 adm2009 039 01_2022 001 100 0 123 SF _adm2009 09 01_2022 003 100 0 3158 adm2003 09 01_2022 003 Eo 3035 adm2009 09 01_2022 002 thy 0 adm2003 09 01_2022 001 KRY 123 Figure 6 79 Left Selecting three data files for grouping Right The resulting grouped files are highlighted 6 15 Export sample list It is possible to export the sample list to an excel table The sample list table is a summary of all samples with corresponding statistics To export a sample list 176 1 Sample on the Sample menu 2 Highlight the sample files that must be exported Right click and select Export sample list File Edit View Mode Analysis Window Help admin Halal elaia eE a e O E E ie Samples Experiment Tools Channels Sample Statistic Celts ooo 0 Figure 6 80 Three files were selected for export 3 Configure the Export sample list box as follows d Export sample list Options Region functions Feature functions Clipboard File Location Public alll Private Name srv2009 02 04_2062 052 ori x
3. File Edit View Mode Analysis Window aga dds doaa ali 1 Owaa Samples Experiment Tools Channels some Staite Ta 0 ae p l na N68 pee 100 14887 a Ea Bor 31 0 13550 f S p2 75 1 SOP 536 7975 c Select the required option s Click Apply and OK Region properties Ce 3 To change region functions a Click the icon which is adjacent to the plot of interest 167 b Select the required options Click Apply and OK ee ee A pa pees ee pei T re A ee i ml am eae E ee ae T Le A ee 4 In the example below region P3 was displayed in blue and set as a NOT region 6 13 4 Post acquisition data analysis Note For an overview of data handling e g opening saving and importing files with the MACSQuantify Software refer to section 6 9 To open saved data files 1 Click File and Open or click the icon H 2 Click the Data files tab eE 3 Choose the desired data file s from a Public Private or External source admin VEE OTE Ee es 168 Figure 6 71 Three MACSQuantify Software data files were opened from a public Shared location Applying analysis templates to post acquired data It is possible to apply or view an analysis template that was used in a previous experiment i e the gating strategy and corresponding plots that are associated with acquired data files can be reapplied and viewed To
4. 1 Click the Keyboard icon 3 Click the Keyboard icon once again to close touchscreen keyboard 5 5 Defining an experiment Note All experiment and analysis templates are defined by the administrator or a Custom user The express user may apply these settings to newly acquired data but can not create analysis templates In order to perform an experiment that the following criteria must be defined 5 5 1 Rack Five different kinds of sample tube racks are available see Table 5 2 for details The Tube Racks Chill 5 Chill 15 Chill 50 and 96 well must be used with the MACS MiniSampler To select a rack configuration perform the following steps 1 Click on Definition to define the experimental setup 2 Choose the rack format using the Rack dropdown list In this example Chill 5 tube rack was chosen for the measurement a wi amp Figure 5 4 Selecting the Chill 5 tube rack for multisample labeling and cell analysis 77 3 For details on how to choose the correct rack format refer to Table 5 2 Rack type Option on Corresponding MACSQuantify Rack MACSQuantify Rack drop down list Graphic Single tube 1 x5 mL Not applicable rack Chill 5 24x 5 mL Chill 15 15x 15 mL 5x5 mL Chill 50 Chill 96 96 well rack 96 microtiter rack plate Table 5 2 Overview of the various rack types that may be used with the MACSQuant Analyzer An appropriate rack should be used
5. Analyzer MACSQuant Running Buffer MACSQuant Storage Solution MACSQuant Washing Solution Bottle closure Column connector Column substitute FITC Fluid container Fluid sensors Fluid sensor cable Front panel MACS Technology Glossary Hydrophobic 0 2 um air filter attached to the bottle closure Used to vent the bottle and simultaneously prevent contaminants from entering or escaping from the fluid bottle Luer to thread connector for attaching the air filter to the threaded bottle closure vent Allophycocyanin Separation column specifically designed for the MACSQuant Analyzer An automated flow cytometer that is also referred to as device or instrument Sterile and ready to use buffer for flow cytometry cell enrichment and washing programs The tubing connector is color coded blue Sterile and ready to use solution for overnight and longterm storage of the MACSQuant Analyzer The tubing connector is color coded black Sterile and ready to use solution for washing and special cleaning programs The tubing connector is color coded green Vented screw on closure with fluid uptake tubes The bottle closures contain fluid sensors and are equipped with sensor cable connectors Each bottle closure is color coded Luer to thread connector holding the MACSQuant Analyzer Column Column devoid of any paramagnetic particles This column substitute can be used when enrichments are not
6. Australia Miltenyi Biotec Australia Pty Ltd Unit 16A 2 Eden Park Drive North Ryde NSW 2113 Australia Phone 61 02 8877 7400 Fax 61 02 9889 5044 macs miltenyibiotec com au Benelux Miltenyi Biotec B V Postbus 85183 3508 AD Utrecht Netherlands Customer service Belgium Phone 0800 94016 Fax 0800 99626 macs miltenyibiotec nl Customer service Luxembourg Phone 800 24971 Fax 0800 24984 Customer service Netherlands Phone 800 4020120 Fax 0800 4020100 China Miltenyi Biotec Shanghai Office Fareast International Plaza A Rm 2301 No 319 Xianxia Rd Shanghai 200051 P R China Phone 86 21 62351005 Fax 86 21 62350953 macs miltenyibiotec com cn 205 Singapore Miltenyi Biotec Asia Pacific Pte Ltd 7 Temasek Boulevard 16 06 Suntec Tower One Singapore 038987 Phone 65 6238 8183 Fax 65 6238 0302 macs miltenyibiotec com sg Japan Miltenyi Biotec K K Nittsu Etai Building 5F 16 10 Fuyuki Koto ku Tokyo 135 0041 Japan Phone 81 3 56 46 8910 Fax 81 3 5646 8911 macs miltenyibiotec jp France Miltenyi Biotec 10 rue Mercoeur 75011 Paris France Phone 33 1 56 98 16 16 Fax 33 156981617 macs miltenyibiotec fr Italy Miltenyi Biotec S r l Via Persicetana 2 D 40012 Calderara di Reno Bologna Phone 39 051 64 60 411 Fax 39 051 64 60 499 macs miltenyibiotec it Spain Miltenyi Biotec S L C Luis Bunuel 2 Ciudad de la Imagen 28223 Pozuelo de Alarcon Madrid Phone 34 9
7. Insert a rewritable DVD into the MACSQuant Analyzer DVD drive Only DVD R or DVD RW media may be used DVD RW and CD media types are not currently supported Wait for 10 20 seconds after inserting the DVD into the drive Click the backup icon located on the top menu bar The files will be written to DVD Note Depending on the amount of data the backup procedure may take several minutes When the progress bar displays 100 the MACSQuantify Software will verify the data once again this may take a few minutes to complete Note At this stage data will NOT be deleted from the MACSQuant Analyzer the data is only copied to DVD 92 6 Insert the backup DVD into the destination DVD drive of an independent personal computer on which the MACSQuantify Software is preinstalled This computer can be used for data analysis 7 Start and login to MACSQuantify Software on the personal computer 8 Click estore Note MACSQuant Analyzer data will be copied to the local drive of the personal computer After a successful data transfer the copied data will be marked as successfully copied on the DVD Note When performing a future data backup on the MACSQuant Analyzer ensure that this backup DVD is used Performing subsequent MACSQuant Analyzer backup 9 Insert the designated MACSQuant Analyzer backup DVD 10 Click backup Note The MACSQuant Analyser software MACSQuantify will identify data marked on the D
8. ccceeeceeeceeccceecueecueeeues 202 13 Tec CIV ICG ccscicsi aes cata ete iectes hot tee ete et eet el 205 14 Ced Warran ON cat hia cepa ih cm endo eet escalate eae EEA 207 15 GOSS ANY shat rac cured eerie Oia he eG aaah oe ne a ahah mane rake kha Thank you for choosing a Miltenyi Biotec product The MACSQuant Analyzer is an innovative instrument for automated multiparameter cell analysis Excite and inspire a Smee RR Sac em Gai Led i Stee SGaeaane tettek y i e ni Fa ERE lt em ft 4 p L a 1 1 1 Important information Please read before use Please read all information contained in this user manual before use Failure to read and follow these guidelines could lead to improper or incorrect use handling or care of your instrument and could cause hazards to users unpredictable results device malfunction or damage premature wear and reduced life time of the instrument and may void your warranty Keep this user manual in a safe place accessible for anyone using the MACSQuant Analyzer This chapter describes the safety instructions and site requirements for your MACSQuant Analyzer The following warnings and cautions are provided to help you prevent injury to yourself or damage to the device Symbols and hazard levels 1 1 1 Setup of safety notices Example Moving parts stay clear Hazard of puncturing or crushing fingers Operate only with attached needle arm guard The safety
9. Create New Plot Window Figure 6 57 Six analysis templates are available to choose from Six analysis templates are available to choose from The size and layout of the dot plot windows are predefined however users can change the properties of each individual plot using the i icon Note To modify these settings during live data acquisition the MACSQuantify Software must NOT be in analysis mode ie All To modify i settings on post acquired data data opened from a file the MACSQuantify Software must NOT be in analysis mode i e Al Custom mode users can select a display format for data and text as follows 1 Click on the icon i located beside the dot plot histogram or text table A popup menu will appear i Dot plot Density plot Histogram Statistic Text Multilayer mode Properties Figure 6 58 Five display formats may be chosen Dot plot Density plot Histogram Statistic or Text JaA 153 2 Select the desired display from the popup menu arv2008 12 10_2063 014 neg 1000 7 San aE E ae eee eee A A SSC A Oo 250 500 1000 FSC A 750 Figure 6 59 Dot plot format was chosen to display the opened dataset aa cick E 3 Optional Click to save changes to the Workspace 6 12 3 Changing the properties of a plot histogram or text table Note The MACSQuantify Software can display data in four discrete formats Dot plot density plot histogram and statistic In addition text
10. Note If a copy of the imported file already exists on the personal computer the following dialog box will appear Overwrite 3 File C cap user adminjdevice Cell cycle analysis cfg exists Overwrite To overwrite a single file click Yes To overwrite all files for import click All No aborts the procedure Note The imported files are copied to MACSQuantify Software It is necessary to delete files off the memory stick using windows explorer Note It is of course also possible to simply move files from the memory stick to a personal computer using windows explorer 6 10 3 To perform backup to network drive 1 Please contact your administrator if a network drive has not been configured for backup Note If a network drive is not configured the MACSQuant Analyzer software MACSQuantify will search for USB and DVD backup media instead 136 2 Click 3 The files will be automatically written to the network drive 6 10 4 Configuring data backup settings administrators only 1 Click Edit Options Software and Backup Options Users Experiment E Instrument B Software Files Keyboard Timers Backup Backup Always data files Acquire DARION Automatic Ask Export H Regions Windows Views Figure 6 36 Changing the default file backup settings 2 Use the dropdown list and or radio buttons to activate deactivate a feature Files Backup a always ask befo
11. User action to select Effect multiple sample positions Single right click of the multiple sample sea Deselect All menu button Clee Al Clear Selected Details Use this button to change the settings for all rack positions Note In order to set all rack positions to allow Measurement and modification of the experiment strategy e g labeling Click Select All 79 Single right click on O Selection deselection of an entire column header TT sample column Deselect Col Clear Col Clear Selected in Col Single right click on Selection deselection of an entire row sample row In this example Row A is selected for sample labeling and measuring Row B is selected for sample measurement only Single right click over Right click over a single rack position a single rack position to completely clear this position In this earal example position A2 will be cleared Table 5 4 An overview of the possible configurations for rack positions 5 5 2 Sample ID and Description The Sample ID and Description boxes are text fields in which alphanumeric characters may be entered in order to name the sample Sample ID and provide a more extensive description Description about the sample The Description field is optional Djela l am amp wo woa Custom Definition Acquisition Analysis Pack O OOO0O0O0O Ci DOO O OL il Description Textfield woe EE SLSLC7e
12. m E Zolfset 0 Start calibration Save calibration Test G H Figure 3 28 Calibration of the 96 well rack is underway The yellow closed circle indicates that the needle arm may be freely moved for manual calibration 5 The needle should be positioned as follows i At the center of well on the y axis i e equidistant from the plate well edges ii Only a fraction of a millimeter from the bottom of the plate well z axis i e almost touches the bottom of the plate To check the needle position relative to the bottom of the plate well gently wiggle the plate to ensure that there is a small amount of movement If this is not the case carefully adjust the needle arm accordingly 6 When satisfied with the needle position click Save calibration 7 A successfully completed procedure is indicated by a green closed circle 52 Calibration of MACS Reagent Rack 4 1 Ensure that the MACS MiniSampler is correctly attached to the instrument 2 3 4 5 This includes fastening of the corresponding cable to the External CAN port located at the back of the instrument see section 3 2 3 for information about correct connection of the MACS MiniSampler Ensure that the MACS Reagent Rack 4 is correctly placed on the MiniSampler Click Start calibration A dialog box will prompt you to ensure that the Reagent Rack is indeed correctly placed and that the single tube rack is removed If this is the c
13. 30 xla 0 xla 5 via i e axi Eaz z E 30 la 1 0 vie 5 vial i c 30 vla Ea Ca fi o xla vd 5 via Li D oxla 01 v 4 Caa fi Figure 6 22 Experiment definition for sample positions Al and A2 Note that reagent vial MC CD14 Monocyte Cocktail human was defined to position one of the MACS Reagent Rack Red box Default annotations yellow were used for the analysis channels 124 4 Rack positions Al and A2 are defined 5 Left click once on the rack coordinate A3 to define experiment settings for this position 6 Define the experiment settings for position A3 as shown below see section 6 3 6 for more details on defining experiments Samples Experiment Tools Channels Experiment Rack Chill 5 tube rack Bo File Tin Ss 001 va O Project MACS Control Cocktail CD14 O Sample ID Samples 1 amp 2 Description Human PBMC Flow rate Low Medium see High Pickup and measure Liquid sensor C Mix sample Mode Standard v Uptake volume 50 pl ij Sample volume 200 p J Annotations Autolabel Settings Custom Express Instrument setting imu EUs inn C Analysis template C Gate C Events 10 000 LJ E 12 3 4 5 6 a 000 Figure 6 24 The sample in rack position A3 will be analyzed in custom mode using a previously saved Instrument setting and Analysis template 7 The samples have been assigned to a rack positions and define
14. DALRA K nen ee Oe Rn ee ee ee ee ee ee ee eee er ee 7 7 D2 SANIDIC 1D and DCS CHOU ON irea aa E etna eis I 80 Io MOG C ar a a oases O E ode nl aa eee E 8l 5 6 WORKING WITH DATA FILES IN EXPRESS MODE ecceceeceececeeceeceeceeeeateateaeenteatecteteateateateaseaeeneas 83 520 1 INTHOCUCTION 10 THE RAN GHG orire E ledcin Genesee evade ner tenad san E R R 83 SOL OPENI TCS oE TE T E A O 84 TOS SAVIO TICS area E E O EO E EE A A 85 5 7 DEFINING AN EXPERIMENT IN EXPRESS MODE A WORK THROUGH EXAMPLE cseceeeeceeeeeeaeeeeaeenvass 86 5 8 READING REAGENTS WITH THE CODE READER IN EXPRESS MODE ceceeceeceeeeeteceecteeeateateateaeeatesees 89 5 O PRINTING IN EXPRESS MODE aveavecveatdcataiessesccacanvucetsseeeceteansie E E 90 5 10 MACSQUANT ANALYZER DATA BACKUP IN EXPRESS MODE cseececceeeeeeeueeeeceeeeeceeeeeueueueueeeneunes 91 5 10 1 To perform a backup tO a rewritable DVD iicicccccccenececenenenscceececesenensneneaeasesesenens 92 5 10 2 To perform backup tO a USB memory STICK cisccccececececcccucecenencenecenencesensnnensnenseneas 94 5 10 3 To perform backup CO network drive wiccececenencncccccccececenensneceaeaseseseneneneneaeaseseseness 95 Sel FLOGGING OUT FROM EXPRESS MODE atsisacscantemnasecacs rar NA aea nid scan anmestonueetsicceedacdacess ea 95 5 12 HOW TO CLOSE THE MACSQUANTIFY SOFTWARE sceceeeeceeeeeeeeecueeuecueeueceeeneueeeeeueeueueeeeeuneens 96 6 CUSTOM ModE eesriie a Ar OA EE 97 6 CUSTOM MODE QUICK REFERENCE GUIDE
15. Figure 3 20 The rack should click into place Figure 3 20 The single tube rack is attached to the instrument as illustrated above 2 Click the Experiment tab and select the Single tube rack format from the Rack pull down menu Note If the adjacent checkbox is activated MJ the rack will be detected automatically 48 Samples Experiment Tools Channels Experiment Rack Single tube rack Oo File adm2009 09 07 001 va O Figure 3 21 Selecting a single tube rack for rack calibration 3 Click Start calibration The needle arm will automatically move forward and insert the needle into the single tube The needle should be positioned as follows i At the center of tube on the y axis i e equidistant from the tube edges ii Only a fraction of a millimeter from the bottom of the tube z axis i e almost touches the tube bottom To check the needle position gently wiggle the tube to ensure that there is a small amount of movement If this is not the case carefully adjust the needle arm accordingly When satisfied with the needle position click Save calibration 4 A successfully completed procedure is indicated by a green closed circle 5 Click Test to confirm that the correct coordinates are saved Needle arm calibration relative to washing station Zoffset 0 gt Start calibration Save calibration Test Home pos 0 00 mm Measure pos 29 10 44 50 mm Zoffset 0
16. File adm2009 09 07 001 vial O Project Ei SamplelD lt Jo Description _ E z Flow rate Low n Medium se High Pickup and measure Liquid sensor C Mix sample Mode Standard f w Uptake volume 150 pl LJ Sample volume 200 pl iJ Annotations Autolabel Settings Custom Express Type Setup i Mode Calibration Figure 3 37 Setting up calibration in Custom mode using the Express option radio button 2 Select the Settings tab in the lower section of the panel and click on the Express radio button 3 Click the Type pull down list and select Setup Similarly choose Calibration from the Mode pull down list 4 Click on the Start Measurement button gt This will start the calibration process Setting the dilution and mixing of the calibration beads prior to calibration Dilution of the calibration beads can be performed by the MACSQuant Analyzer 1 Select the Autolabel tab within the Experiment tab 2 Click lt add gt This will introduce a Reagent dialog box 3 Select S1 and prebuffer and adjust the dilution appropriately The buffer should be set at 10 1 with no incubation time 59 4 Follow the steps described for manual calibration 3 7 Compensation of the instrument settings A proper compensation of the instrument is crucial for the optimal acquisition and display of data obtained by flow cytometry Compensation essentially accounts for the inherent o
17. Miltenyi Biotec Asia Pacific Pte Ltd Singapore Phone 65 6238 8183 macs miltenyibiotec com sg Miltenyi Biotec S L Spain Phone 34 91 512 12 90 macs miltenyibiotec es Miltenyi Biotec Ltd UK Phone 44 1483 799 800 macs miltenyibiotec co uk i S E h
18. Print Ctrl P Save Click to save Workspaces Instrument settings Experiments Reagents and Eh Print al Ctrl Shift P Analysis templates depending on user access rights set by the administrator oga Logout g y Import FCS file Import data files in the FCS file compression format Import Import data files from an external media source e g external hard drive Export Export data files to external media e g external hard drive Print Print a selected area Print all Print the entire workspace Logout Logout of the current session Edit Edit View Mode Analysis Window Help o Command Description Undo _trl Z Undo Redo Undo or redo the most recently Re performed action e g undo create Copy page Ctrl C region RA Copy plot Ctrl Shift C Copy page Copy the entire analysis window to the x clipboard All dot plots and tables are Delete region Del i copied Elli gt Br Copy plot Copy a single selected plot histogram _ Rectangle highlighted in green C_ Polygon Delete region Delete a region that was created in a dot plot Quadrant Ellipse Rectangle Create the afore mentioned geometric Interval Polygon Quadrant shape ina plot G User settings Interval E Instrument settings Ctri Alt I User settings Create and or modify user account settings Administrator only Rack Ctrl Alt R g Instrument Modify the instrument settings E Reagents i a Settings comprising Channel Comp
19. Users Experiment E Instrument Software Keyboard Acquire E Export Backup Timers Standby timer 120m min 0 min LA Shutdown timer Needle priming time H Regions E Views Shutdown default behavior Analyse mode Instrument off Figure 6 46 Changing the default timer settings 2 Use the slider bar or text field to change the timers for the Standby timer instrument goes into standby after the stipulated time of inactivity Shutdown timer instrument performs a controlled shutdown after the stipulated time of inactivity Needle priming timer instrument performs an automated needle priming at stipulated intervals 3 Shutdown can be defined as Instrument off default or Analyse mode the instrument switches to Analyse mode and does not power off 4 Click Apply to implement changes Click OK close the window 5 Click Edit Options Software and Timers To assign default settings for Acquire Note Feature only applicable to the MACSQuant Analyzer This function is not available to MACSQuantify Software installations on personal computers Options H Users Experiment E Instrument Software Keyboard Timers Export Backup H Regions Views Acquire Reset sample on clear view o v lt l gt Live events C Export as FCS Show volume progress in pl Apply express analysis Figure 6 47 Changing the default Acquire settings 144 2 Use the
20. and backed up in Custom mode Data files may be stored to and therefore opened from a Public Private or External file location 127 e Public files are located on the local hard drive of the MACSQuant Analyzer or personal computer and are accessible by all users e Private files are located on the local hard drive of the MACSQuant Analyzer or personal computer and are only accessible by the logged in user account e External files are located on an independent file storage device which is connected to the MACSQuant Analyzer or personal computer via the USB port i e a memory stick The default window for saving and opening data files is composed of the following tabs Figure 6 28 The default window for opening and saving various file types Note The availability of these tab options is dependant on the user profile Custom user Express user or administrator and whether data settings are being saved or opened Tab option Description The Workspaces tab allow users to save user an entire workspace which is composed of instrument settings experiment and reagent definitions and an analysis template with accompanying data Instrument settings are compensation and calibration parameters for the MACSQuant Analyser These parameters are important for data analysis and are vital to maintain standardized results over time and from instrument to instrument The MACSQuantify Software can open and save instrument settings
21. depending on the sample number and volume Configuring the sample rack 1 Click on a sample position using the left mouse button This will allow you to select deselect and activate deactivate the sample Refer to Table 5 3 below for a summary of the potential rack configurations for single sample positions 78 User action with left mouse Effect button or fingertip action on touch screen Single click on circle single finger touch Double click on circle double finger touch Details Default open circle indicates no operation Clear Closed green circle with orange rim Sample selected for measurement Orange circle indicates that the sample is activated and any alterations made to the measurement Strategy e g labeling will only apply to sample positions with this designation Closed green circle Sample selected for measurement Closed blue circle Measurement in progress Closed gray circle Measurement finished Closed yellow circle Processing of sample has commenced e g sample has been labeled and incubation is underway Table 5 3 Summary of rack configurations 2 Use the right click button to select deselect single or multiple sample positions The entire rack may also be selected deselected or even cleared using the multiple sample menu button located at the top left hand corner of the dialog box Rows or columns can also be selected or deselected by clicking on the letter or number respectively
22. located in the center of the fluidics system and has a slot for the insertion of the MACSQuant Column Sample fraction containing the cells not magnetically labeled During MACS Separation these cells are not retained on the column and pass directly through the column while the column is in the presence of the MACS magnet R Phycoerythrin Sample fraction containing the cells labeled with MACS MicroBeads after MACS Separation These cells are retained on the column while the column is placed in the magnetic field The cells are eluted from the column after the column has been removed from the magnet Process of cell enrichment is accomplished by the MACS Cell Enrichment Unit and the MACSQuant Column Target cells labeled with MACS MicroBeads are enriched using MACS Technology Computer controlled high precision syringe pumps with Teflon seal plunger that drive fluids through the MACSQuant Analyzer fluidics system High resolution TFT color touchscreen located on top of the MACSQuant Analyzer The touchscreen is used to operate and monitor the instrument via On screen menus Threaded plastic connector with a square nut used to connect the tubings to the bottle closures the column the pump or valves Permanent set of Teflon tubing through which fluid circulates in the MACSQuant Analyzer fluidics system Different acrylic tube racks are available with the instrument and are designed for optimal positioning
23. sasrioinonsanr ni a vine seveeaaNe wees 97 6 1 1 Quick guide to the top menu bar ICONS vicucccecencncccenececcetensasenensceesenensesenensesenensnsets 97 6 1 2 Quick guide to the MACSQuantify Software Menus wiscccenenencccccecerenerenenscsasesesesenens 99 OZ USER ADMINISTRATION orna a E E a 101 07 i Creanga NOW USEF soine o a died 101 6 3 GETTING STARTED IN THE CUSTOM MODE accainn E eee 103 O TUT OW TMC ISU UICC a E O a E 103 6 3 2 Login as administrator OF CUStOM USEf sisacccccccecenecensnenesenenesenenensnsananesesenensnanenes 103 6 3 3 Activate the touchscreen keyboard on the MACSQuant ANAIYVZEP ssseccnccsecenensecens 104 O33 SF CHECK TNOTIVUIG JOVEN S iia eae o etter asta eee oie et cha ge eae hie atoeeee 104 03S CHECK TNE TNS OOMEN STATUS duce hati ieee ad E tech edi ake 105 6 3 6 Define an experiment in Custom mode wiasesecececenenenencccceseseseneneneceauasesenenenenenenes 107 6 4 SWITCHING TO EXPRESS MODE FROM CUSTOM MODLE cecceeeceececeacececeeceaseaeeeeaeeseaseaeaeeataneas 113 6 5 PRINTING IN GUSTOM MODE coracana iebaticsi i acoiend a eee cca 113 6 6 REAGENT MANAGEMENT c2scctccutvass icestee iin beh a ocoet eie alanine d eeaee cee 114 6 6 1 An overview of the MACSQuant Analyzer reagent management windowWw ELS 6 6 2 Selecting and ASSIGNING reagents ITIANUANY ci scccccenenenenececeesecenensnensnsnsasenenesenenenes 116 6 6 3 Scanning reagents With the 2D code LCALCL scscccenencnenencncecenenensnensnsas
24. 136 6 10 4 Configuring data backup settings administrators OPV visicccnenenenerececesesesesenens 137 6 10 5 Configuring network settings administrators only cescccccceneneneneneneneneretesesesesess 137 6 1 1 CONFIGURING THE DEFAULT USER INSTRUMENT AND SOFTWARE OPTIONS cccceeeeeeeeeeceeeceeeeatens 138 6 1 li Changing the default user ODUONS ici eicsts aise en a de 138 6 11 2 Changing the default experiment OPCiONS ccccececcccececenensnensnenenenenecesesesesesesasans 14 6 11 3 Changing the default instrument options uasecececccencnececeececeserensnenenseeecesesesenenenes 141 6 11 4 Changing the default software Options essceccccccececenenenenensnenenenenenesesesesesesesesasans 143 6 12 DATA ANALYSISIN CUSTOM MODE ssseiiceiscosoap raias or eua area e EAT Erabi iS denai 151 6 12 1 Creating a new analysis template or analysis WiNdoOW uusseseneceecerecseserersesesensnsess 152 6 12 2 Choosing a display format for plots and PISCOGIAINS vicsecececenenenenensnseceseserenenenes 153 6 12 3 Changing the properties of a plot histogram or text table wiscscccccecenececeseceseeess 154 6 13 WORKING WITH REGIONS OR GATES ccecscscrevsccesecsececucrevsesesecsececucravsscusessecacuararsssusessecas 160 OF S10 DIAG FOGIOIIS ivi esi eera ENEE AAEE EEEE E AEA EREE EEEE 160 6 1532 GAGNG S WAT OS nas tosnrstihces Get orsadbnn A ison spun iin AA EEE REREN 165 6 13 3 Changing the properties Of regions viccecececeececceecenensnsnsnenenenenetesesesesesesesesen
25. 2 Air circulation Ambient air temperature might not be adequate to cool the MACSQuant Analyzer to acceptable operating temperatures without adequate circulation Make sure that the room in which you operate the instrument has adequate air circulation The instrument 14 should not be placed next to radiators heat registers stoves or other pieces of equipment including amplifiers that produce heat Allow sufficient air circulation around the MACSQuant Analyzer at least 15 cm on all sides during operation to ensure adequate cooling of the instrument Prevent direct exposure of the instrument to sunlight Slots and openings of the instrument are provided for ventilation and should never be blocked or covered as these ensure reliable operation of the MACSQuant Analyzer and protect the device from overheating Never push a foreign object through an opening into the instrument 1 4 3 Water and moisture Do not use the instrument in a wet or damp location Avoid high humidity or condensation and protect the machine against water splashes 1 4 4 Grounded earthed product The instrument is equipped with a three wire electrical grounding type plug that has a third pin for grounding This plug only fits into a grounded power outlet This is a safety feature Do not try to insert the plug into a non grounded power outlet If you cannot insert the plug into the outlet contact your local electrician to replace the outlet 1 4 5 Power sources T
26. C Laser 405 nm 25 C Note Base plate temperature for 488 nm laser 10 45 Note Bench temperature 33 C Note Fan speed This will fluctuate automatically depending on the ambient room temperature and internal temperature of the MACSQuant Analyzer Note If errors are reported please refer to section 10 troubleshooting or Miltenyi Biotec technical support 11 1 2 Sample uptake The Sample uptake tab indicates the status of the robotic arm as well as whether the MACS Mini Sampler is connected or not The relative position of the needle arm is listed in the lower left box and if the single tube rack is connected 192 E Hardware monitor MacsQuant SN 23 x Fluidics Lasers and detectors Camera System Sample uptake needle rns ampler p Fos 2 mm 2 Pos 0 mm SingleRack connected Figure 11 2 Real time hardware monitor of the sample uptake arm Name Further information Sample uptake needle Indicates the coordinates of the needle schematic arm MACS MiniSampler monitor The MiniSampler is not connected Liquid uptake rates Measuring mode Sheath pressure Buffer Sheath buffer Total volume Total time sec mbar consumption consumption uL for rinsing uL during rinse cycle uL 4166 6 4916 6 Standard 4875 1700 6575 Extended 5250 5800 11050 72 Sheath consumption for a 100 uL measurement without rinsing volume Measuring mode Sheath pressure Measurement Sheath buffer consumption during Total mbar and
27. Experiment definitions for this sample can also be modified using the Experiment tab e g sample name labeling strategy uptake volume etc b designed sample will be measured and associated Experiment definitions can not be changed 3 Use the Experiment tab to change the Experiment Flow rate and Pickup and measure options as required Note To reiterate Only sample positions with status can be programmed using the Experiment tab Note For more information about defining an experiment refer to section 6 3 6 Define an experiment in Custom mode 4 To close the popup rack window click the Rack icon 5 Before starting the run check that a Experiment definitions are correctly assigned to each rack coordinate and that each sample is correctly positioned on the Chill Rack b Sufficient quantities of reagents and buffers are provided Ensure that the waste bottle is empty c The reagents have imported and assigned to a position on the MACS Reagent Rack see section 6 6 d The instrument is correctly calibrated and compensated see sections 3 6 and 3 7 6 8 Defining an experiment with multisample processing A work through example 6 8 1 Background In the following example CD14 cells from three human PBMC samples were enriched using the autoMACS Pro Separator in combination with the Monocyte Isolation Kit Il It was important to evaluate the outcome of these cell separations using flow c
28. Get si 1i 15i Ant L iT PE A ete eee Be La og Pues ae EOS aA CM Firc a 1e Tee 1 et 1 Set Tiit 12e Tisd 101 12 1 21 Ti 12e ri T7 PE A ob L 17 PE A Page 11 Figure 5 20 Half size example print out of data analyzed by the MACSQuantify Software in Express mode 5 10 MACSQuant Analyzer data backup in Express mode It is recommended that data is regularly backed up to an external location Data can backed up to a network drive USB memory stick or DVD Administrators can configure data backup settings Please contact your administrator for more information or refer to section 6 11 4 of this help guide Note Before performing Backup ensure that the desired backup media is accessible to the MACSQuantify Software 91 Backup media The backup procedure searches for backup media in the following order 1 A designated folder located on a local area network this must be setup by an administrator with assistance from Miltenyi Biotec technical support 2 Amemory stick attached to the USB port on the MACSQuant Analyzer 3 Arewritable DVD 4 If none of the above are found the MACSQuantify Software reports an error No valid backup devices found 5 10 1 To perform a backup to a rewritable DVD 1 2 3 4 5 Ensure no USB stick is installed and that no network drive has been defined as the default location for backup files Please contact your administrator for further advice
29. It is assumed that the instrument hardware and settings have been correctly calibrated The analyzer should have been correctly compensated 13 Click Start Measurement LJ to start acquisition Table 6 4 shows an overview of the above described procedure for defining an experiment Samples Experiment Tools Rack Rack Chill 5 tube rack 7 E File adm2009 05 12 007 xla Experiment Sample ID Sample 1_CD14 analysis d Description MC CD14 Monocyte Cocktail fl F Flow rate Low Medium sae High Pickup and measure Liquid sensor Mix sample Mode Standard v Uptake volume 100 pl Sample volume 200 pl c Annotations Autolabel Settings Custom Express Instrument setting CD14 Experiment 1 v Analysis template CD14 v C Live gate C Events 10000 4 0060 B E D m irni ar e a Sethnge fy MIC CD14 CI iad CI ca C cack Cl cak E cack C cack Cl cag C cak ar Selected setting Chill 5 tube rack File name adm2009 05 12 001 Sample ID and Description Flow rate Mix sample checked activated Custom mode Events Rack configuration Autolabel tab Description A chill 5 tube rack will be used for this analysis Filename is automatically generated To enter a name manually uncheck the associated box to the right The Sample ID and Description are text fields entered by the user Medium flow rate was selected 50 L min The sampl
30. SS SS SY Samples Experiment Tools Channels H Coitate uptake unit Uptake unit calibration Calibrat detection 1 First push the Start calibration button to begin the calibration be careful the hardware moves 2 Move the needle and sampler by the hand to the specified position and press Save calibration 3 Test your calibration using the Test button select position you want to test Remove external media Needle arm calibration relative to washing station Zoftset 0 S Start calibration Save calibration Test Home pos 0 00 mm Measure pos 29 10 44 50 mm Single tube rack Z offset 0 S Start calibration Save calibration Test Offset 64 90 51 10 3 30 mm Chill 5 tube rack Zoffset 0 gt Start calibration Save calibration Test Offset 13 60 44 70 0 10 mm 96 well Z offset 0 S Start calibration Save calibration Test Offset 13 90 45 30 4 00 mm Reagent Rack 4 Zoffset 0 Start calibration Save calibration Test Offset 11 60 45 40 4 00 mm Arse Date analysis mode Time Yokas 10 56 oO A Ol Figure 3 29 All uptake units have been correctly calibrated If 53 3 5 2 Calibration rack detection Single tube rack and acquisition button detection 1 Click on the Tools tab and Calibrate rack detection 2 Click Start calibration The dialog box will prompt you to remove the sing
31. These settings can be applied to acquired data and thus this useful feature allows users to perform recompensation after data acquisition The Instrument settings may be saved but not opened in Express mode Experiment definitions can be saved for future use Reagent type and corresponding Reagent Rack 4 positions sample rack type and corresponding Chill Rack sample positions the analysis mode and sample processing definitions e g labeling strategy comprise experiment definitions Reagent type and position on the reagent rack can be saved using the Reagents tab The Reagents tab is not available in Express more Analysis templates are predefined analysis layouts for data acquired by the MACSQuant Analyzer The templates are creating by defining a gating strategy with associated plots histograms tables and statistics Administrators and Custom users can customize and save templates for reuse Express users cannot create or modify Analysis templates Data files can be saved to a Public Private or External file location by all users MACSQuant Data Data MQD is the standard file handling format however the MACSQuantify Software can also import Flow Cytometry Standard FCS file types 128 6 9 2 Opening files 1 Click to open the Open window Figure 6 29 Custom mode users and administrators are able to open Workspaces Instrument settings Experiments Reagents Analysis templates and Data files 2
32. Viable CD14 monocytes pt2 00 Oe 2 1 0e 21 0e 11 0e0 1 0e1 1 0e2 1 0e3 Figure 6 72 Application of an analysis template for the file EEEVEAKAUE uE The template shows a gating strategy for analysis of CD14 monocytes 169 5 To View analyzed results of the previously defined analysis right click on the data file and select View with analysis lt name of analysis gt le Edit Mi Analysis Window Help Open Ctro dBal x P Add Samples Experiment Tools f Import FCs fie Sample Statistic Cel live 0 0 H Export Sample st 2008 12 10_2063 015 pos 100 0 3 OPI 91 0 Remove SO P2 75 1 2 OP3 53 6 Recompensate OP4 523 gt stv2008 12 10_2063 014 neg 100 0 t 2008 12 10_2063 013 0r1 100 0 Apply instrument settings Apply analysis template Remove all Recompensate all Export sample list admin x ES Ag Dy Be Om e Ss te Se ee O e2 mr 1 0e 21 0e 11 0e01 0e11 0e21 0e3 1 0e 21 0e 11 0e01 0e1 1 021 063 VioBlue A PEA st 2008 12 10_2063 015 pos 108 12 10_2063 015 pos Legend i Region Population Count lt Pl Debris exclusion 1 amp P1 P2 Viable leukocytes 11178 PI P2 P3 Granulocyte k 7977 PI P2 P3 P4 CD14 Monocytes 7730 stv2008 12 10_2063 015 pos Results pos 16 log Freq Conc 17ml Viable leukocytes worm 82 5 2 24E 005 i Viable CD14 monocytes wra 69 7 1 56E 005 1 0e 21 0e 11 0e0 1 0e1 1 0e21 0e3 VioBlue A Fi
33. YSIS aei EATE R A E 21 2 2 MAGS COQUOLAPPhCAt ONS sicnsistunisiiursdesdedoatecusarsticadseeiadscdabseaeneiouserentetnstateateen 21 Poe RUE CCI OCTOCUON oiadi ea TE N E E OE 22 227A ADSOIULC cell guantitatiOl scierie a i E TR 22 2 2 5 Automated cell labeling and ANAIYVSIS iscccceceneneneneneneccenecunenenenscsanesestenenenensnensanaees 22 2 2 6 Flow cytometry an introduction vaucasenenecencenececueneneacensnensanensauanensneananensasanensanansees Ze 2 2 7 Displaying flow cytometric HATA ss ecenencnccencccccecenecenensnsaeaeeenserenensnenenensasenenenenenenenees 24 2 2 8 Analyzing flow cytometric data using regions OF gating sessesssanoannannannenennnn 28 2D MACS C FLE SEPARATION eisian oi o a Ta 29 2 4 DESCRIPTION OF THE MACSQUANT ANALYZER cceececceeeeeceeeeeenceeeeneeeeueueeueeueeueuueeeeuneeeeuneeneas 3 3 Assembly and installation of hardware cccccccesececeseceaeeeeeeeuavaveeeeueuavanenenuaeas 32 3 1 UNPACKING THE MACSQUANT ANALYZER cccccceecceeeeeeeeeeeeeeeceeeeaeeegueeeueeusauenueeunueengueenanenauss 32 3 2 INSTALLATION OF THE MACSQUANT ANALYZER cccceeeceeeeeeeeeeeeuceueeueueeeueueeeeeueeueuneeeeuneennunes 34 3 2 1 Connecting the fluid containers and fluid sensor cables wissisccccneneneneneneneneneceseseces 35 Bee CONMNCCHNG UIC DOWE COO noore orsera ate tatiana E OAA N 38 3 2 3 Installation of the MACS MiniSampler and tube racks and reagent ack ccscevenee 38 3 2 4 Positioning of cooling tube rack
34. a E ET 187 9 1 9 Decontaminating the MACSQuant ANALYZED useccncccecenensenecensesenenscsetensnsenensnsasets 187 10 Troubleshooting sasra a a E 188 10 1 PROBLEMS NOT INDICATED BY ERROR MESSAGES ceccececeeeececceceececeaceceaseaeeseaceataeeanseeaeeneas 188 IOT L OUMINI O iint A AE etd EE E O O AA A 188 lO T2 FPUMmOSV Nge Jak AOC sr anei E E O N EONO A N A 188 EOD AWS Nng Ss aN OV ETT OVE 2 iets Beast tinea tia eee eas vnc ada T 189 10 1 4 MACS MiniSarnpler does NOt move POD CST usacccencceecenececeecenscsetenscsesenensetenenees 189 10 1 5 Air bubbles during measurement or no events are acquired ssssececeneneneneneneees 189 10 1 6 Excessive debris is present in acquisition vacacenccuenccuensenensenensnnensnnenuenenunnennensnses 189 VOLS TOUGISEl CEN Temal S OK nasen dias E EOE E A E 190 11 HardWare MONITO cerirorens uis ae E A ETN Ra 191 Tie HARDWARE MONITOR WINDOW 225uccbacatvnsecnet oecauackehet E 191 llel st TUGCE COMPONETS soraa O E E a aameaseae 191 PUT SPE UDANE AEE enact tases es ae AN BW sg OPI UOO a PR en nn e eRe Te eee E A eee Et RE ne ate Pee ee Ta 193 12 Technical data and Specifications ccccscseseseseseeceeeeeeececeeeeeeeeeeeeeseseseseeeees 195 12 1 LABELED DIAGRAMS OF THE MACSQUANT ANALYZER ccccceeeeeeeeeeeeeueeucceeeeeueeeueueeeeeeeeeeeneens 195 12 2 TECHNICAL DATA AND SPECIFICATIONS OF THE MACSQUANT ANALYZER cccceeeceeeeceeueeeeeueuees 200 12 3 TECHNICAL DATA AND SPECIFICATIONS OF THE MACS MINISAMPLER
35. a first step surface antigens are magnetically labeled in a highly specific manner with monoclonal antibodies coupled to MACS MicroBeads MACS MicroBeads are superparamagnetic particles of approximately 50 nanometers in diameter comparable to the size of a virus MACS MicroBeads are not known to alter the scatter properties of cells in the flow cytometer or influence the light microscopic appearance of the cell They form a stable colloidal suspension and do not precipitate or aggregate in magnetic fields MACS MicroBeads are composed of a biodegradable matrix made of iron oxide and polysaccharide hence it is not necessary to remove them after the separation process saving hands on time MACS MicroBeads do not alter the Structure function or activity status of labeled cells and they are not known to interfere with subsequent experiments Finally MACS MicroBeads offer an extremely flexible tool for the pre enrichment of many cell types from many species through the coupling of different antibodies Several hundred reagents for the isolation of human mouse rat and non human primate cells as well as reagents for indirect labeling of many other cell types are available After magnetic labeling the cells are passed through a MACS Column placed in the magnetic field of a MACS Separator Non labeled cells flow through and can be collected labeled cells are retained in the column and can be released after removal of the column from the magnetic
36. apply an analysis template associated with a data file 1 Open the desired data file s 2 Click on the Sample tab menu 3 Right click on the data file and select Apply analysis template Samples Experiment Tools Channels Sample Statistic Cells live 0 0 0 stv2008 12 10_2063 7 Open stv2008 12 10_2063 Add Rs Import fi F export Sampl Ungrouy Resample Remove Recompensate Apply instrument with Anal 4 The data file and corresponding analysis template will be loaded in Analysis mode Al File Edit View Mode Analysis Window Help admin lam H B X 5 Help Epress Logout d Samples Experiment Tools Sample Statistic Cells tiie 1 2 10_2063 015 pos 1 2 10_2063 01 poe ps 1 2 10_2063 015 pos live 0 0 0 1 BEE ic PAPARE 100 0 14887 750 j SGP 91 0 13552 F Sch p2 1 11178 S 0 lt 3 SIOP 535 781 8 a O PA 51 0 7597 250 stv2008 12 10_2063 014 neg100 0 18222 S srv2008 12 10_2063 013 ori 100 0 22130 ES o 1 pare oe ae eas 1 0e 21 0e 11 0201 0e11 0e21 0e3 0 250 500 750 1000 1 0e 21 0e 11 001 0e11 0e21 0e3 i FSC A PE A 1 sr 2008 12 10_2063 015 pos 1 2 10_2063 015_pos Legend j Region Population Count P1 Debris exclusion P1 P2 Viable leukocytes P1 P2 P3 Granulocyte exclusion PI P2 P3 P4 CD14 Monocytes 1 sr 2008 12 10_2063 015 pos Results neg 16 log Freq Conc 1 ml Viable leukocytes 2p1 100
37. are presented as dot plots histograms and as a tabulated summary on a two page two screen report Successful calibration for each channel is indicated by a green checkmark To view all 57 calibration dot plots and histograms click Next screen or Previous screen File Edit View Mode Analysis Window Help aida ee e esa alt b uaaa Rack eee e J File adm2009 03 01_2022 003 HR Sample ID File Edit View Mode Analysis Window Help dga Le pelea i Oelelele asia Rack Single tube rack L C File adm2009 09 01_2022 003 SJA Project jo Sample ID a Acquisition mode ear T Calibration ok 1 day old Figure 3 36 Successful calibration of the MACSQuant Analyzer as shown by an array of histograms upper and associated summary table lower 58 3 6 2 Performing manual calibration Custom mode users and administrators can perform manual calibration as follows Note The calibration beads must be pre diluted and mixed before performing this procedure The MACSQuant Analyzer can perform pre dilution and mixing of calibration beads see section Setting the dilution and mixing of the calibration beads prior to calibration for more details 1 In the Custom mode select the Experiment tab on the left side of the screen Samples Experiment Tools Channels Experiment Rack Single tube rack O
38. assign user permission to user groups 1 Click Edit Options and Access Options Users Files Experiment E Instrument amp Software Access Public Private Instrument Settings Read Read amp Write Experiments Read Read amp write Reagents Read Read amp Write Analysis Read Read amp Write Data files Read Read amp write Figure 6 40 Changing default file paths for user groups 2 Use the dropdown list for each access category to modify the read and write privileges as required 3 Click Apply to implement changes Click OK close the window 140 6 11 2 Changing the default experiment options The default Experiment settings can be modified using the Experiment options menu To assign default Experiment settings 1 Click Edit Options and Experiment Options Users Sadat E Instrument E Software i Flow rate Medium C Mix sample Standard v Uptake 100 p Volume 200 pl C C Event limit 10000 Figure 6 41 Changing default experiment settings 2 Modify the default values for Flow rate Mode Uptake Volume and Event limit as required Note It is not recommended to activate the Event limit setting LJ Evert limit Limiting data acquisition to the number of events prohibits volumetric cell counting 3 Click Apply to implement changes Click OK close the window 6 11 3 Changing the default instrumen
39. being performed or for long term storage and shipment The column substitute cannot be used for cell enrichment prior to analysis Fluorescein isothiocyanate 1 5 L bottle that holds the system fluids for operational use of the MACSQuant Analyzer Fluid sensors monitor the fluid levels in the containers for Running Buffer Washing Solution Storage Solution and waste via electrolyte conductivity These sensors measure electrolyte conductivity and is integrated into the closures of the Running Buffer Washing Solution Storage Solution and waste fluid containers Cable connecting the fluid sensor to the MACSQuant Analyzer The sensor cables are color coded blue for Running Buffer green for Washing Solution black for Storage Solution and red for waste The front panel opens sideways giving access to the MACSQuant Analyzer Column pumps valves washing station and tubings Technology for immunomagnetic labeling and subsequent separation of cells or biomolecules in a high gradient magnetic field 208 MACS MicroBeads MACS Cell Enrichment Unit not in order Negative fraction PE Positive fraction Cell enrichment Syringe pumps Touchscreen Tubing connector Tubing system Chill Tube racks Uptake needle Waste container Superparamagnetic particles conjugated to antibodies for magnetic labeling of cells or biomolecules Black cover surrounds the area enclosing the magnet The magnet cover is
40. check boxes to activate a feature during data acquisition live mode Reset sample on clear view plot or chart is automatically cleared after data acquisition i e it is not necessary to click the Clear icon E Export as FCS acquired data is always exported as FCS Show volume progress in uL during sample uptake and processing the MACSQuant Analyzer will continually report changes to the sample volume in the Status Bar Apply express analysis The option to perform analysis in Express mode is always given 3 Click Apply to implement changes Click OK close the window To assign default settings for Statistic Export 1 Click Edit Options Software and Export and Statistic Options Statistic Experiment E Instrument Software Destination Keyboard Clipboard File Timers Acquire Conversion Export C Convert decimal point to comma Fes Backup Regions C Reverse sample order Views C Transpose Figure 6 48 Changing the default Statistic export settings 2 Use the check boxes to activate a feature Clipboard plots and data tables can be copied to Windows Clipboard File plots and data table can be exported to another file Conversion activate the following conversion is options for file export a Convert decimal point to comma the decimal point is converted toa comma b Transpose Data is exported as a table comprising rows and columns When Transpose is checked the table format is
41. connections using a wrench 3 Ensure that the fluid containers are filled and installed correctly that the correct tubing and fluid sensor cable is attached to the corresponding container and that the bottle closures are fastened Make sure that the waste bottle is empty Note When working with biohazardous samples it is recommended to fill the container with 100 mL of disinfectant before use e g MACS Bleach For proper disposal follow local regulations 4 Optionally Open the front door and check that the MACSQuant Column is installed correctly Ensure that the tubes are securely fastened to the column and that no part of the visible tubing is pinched or obstructed 42 5 When all points of this installation checklist have been fulfilled close the front door 6 Switch on the instrument using the power switch and pressing the touchscreen by hand 3 3 Materials required for operation of the MACSQuant Analyzer The following section outlines the materials and consumables required for operation of the MACSQuant Analyzer 3 3 1 Buffers and solutions Running buffer washing solution and storage solution are required for daily operation of the instrument Only use buffers and solutions supplied by Miltenyi Biotec for operation of the MACSQuant Analyzer A reproducible and optimal performance of the MACSQuant Analyzer cannot be guaranteed when the instrument is operated using self made buffers and or solutions pro
42. field Thus both labeled and non labeled cell fractions can be efficiently isolated with MACS Technology The entire procedure is fast easy to handle and gentle on cells leading to the enrichment of cells that can be immediately analyzed The MACSQuant Analyzer is equipped with the MACS Cell Enrichment Unit in order to provide a fast and easy way to analyze a rare cell population using MACS Technology The MACSQuant Column when properly seated in the MACS Cell Enrichment Unit can isolate up to 5x107 magnetically labeled cells Upon separation of the magnetically labeled cells the negative and positive fraction can automatically be analyzed If pre analysis enrichment is desired cells can be labeled with MACS MicroBeads separated using the MACSQuant Column and then directly analyzed by flow cytometry in a fully automated fashion Pre enrichment of target cells by MACS Technology before fluorescence cell analysis is particularly valuable when target cells occur at extremely low numbers such as stem cells or antigen specific T cells 30 2 4 Description of the MACSQuant Analyzer The MACSQuant Analyzer is a state of the art benchtop cell analyzer for highly sensitive multicolor flow cytometry Three lasers combined with powerful MACSQuantify Software make for a fast and simple analysis of cells The instrument is equipped with two scatter FSC SSC and seven fluorescence channels Thus the MACSQuant Analyzer is ideal for MACS C
43. filter in the end This end must be in the upward position in order to achieve the best enrichment See section 9 1 3 for more details 5 Align the column so that the top column connector sits on the guide of the magnet cover Press the column into the slot until you feel the guides click Verify that the column is placed in the center of the magnet cover 6 Close the front door 3 2 7 Installation of the webcam A webcam is provided for use with MACSQuant Live Support See section 8 for details of how to contact Miltenyi Biotec technical support via MACSQuant Live Support Note A web cam should be supplied with the MACSQuant Analyzer If this is NOT the case please contact your nearest MACSQuant Specialist 4 To install the webcam 1 Place the webcam in the holder assembly at the side of the instrument Figure 3 15 Location of the web cam holder assembly 2 Attach the camera USB cable to a free USB port at the back of the MACSQuant Analyzer Note The web cam will automatically install If this is not the case please contact technical support or your local MACSQuant Specialist 3 2 8 Installation checklist The following checklist can be used to ensure that the MACSQuant Analyzer is correctly installed 1 Ensure that the power cord is securely plugged into the MACSQuant Analyzer and to a functional main power supply 2 Ensure that all other tubing connections are fastened If necessary tighten loose
44. flow volume uL measurement cycle i e without measurement rate uL min rinse cycle uL time min Standard 200 18000 4 Low 25 ul min Med 50 ul min High 100 ul min 11 1 3 Optical bench Within the lasers and detectors tab it is possible to monitor the status of each laser fluorescence channel and detector The temperature fan speed PMT voltage and 193 annotated path of each laser is shown Fluorescence channels that are active are highlighted A status overview of the optical bench is schematically represented E Hardware monitor MacsQuant SN 23 Camera System Extended info Bench temp BEL Power supply temp 0 18 C Fan speed level 24 Heater state not avail 4oorm C Figure 11 3 Real time hardware monitor of the fluid components 194 12 Technical data and specifications 12 1 Labeled diagrams of the MACSQuant Analyzer The key accessible components of the MACSQuant Analyzer are schematically shown in figures 1 to 5 and can be roughly grouped into six categories comprising e the integrated computer and touchscreen e the robotic arm and sample uptake port e the access cover to tubing system the fluidic system including tubes and buffer bottles e the fluid containers and their holders e the MACS MiniSampler and tube racks e the plugs connectors and guides Integrated computer for control of cell processing All interactions with the computer may be performed using the TFT co
45. gating strategies can be saved for future use as Analysis templates To perform stop gating 1 2 3 4 5 6 7 Click to open a new analysis window Choose the required plot design Create New Plot Window Define the Experiment settings as required e g uptake volume sample name flow rate labeling strategy etc Enter the required number of events for the associated Stop gate Events TOOO0 L Check the Gate option and select the desired Stop gate from the drop down list Annotations Autolabel Settings Custom Express Instrument setting CD14 Experiment 1 w Analysis template Cell Cycle odora cells _ KJ Gate Es P1 v Events 10000 LJ Ensure that the correct instrument settings are loaded and that compensation is correctly performed Note See section 3 6 for information about performing instrument calibration see section 3 7 for information about performing compensation Ensure that enough sample reagents and buffers are provided Click on the Start Measurement button gt The MACSQuant Analyzer will commence sample uptake and measurement 174 8 Draw regions on the plots as described in section 6 13 9 Save the Analysis template for future use 6 14 Grouping data post acquisition What is the benefit of sample grouping The maximum sample volume that can be acquired in a single step by the MACSQuant Analyzer is 450 uL There are occa
46. inverted i e columns become rows and rows become columns c Reverse sample order When exporting sample lists samples can be saved to an excel table in descending or ascending reverse order In the following example exporting the sample list with Reverse checked will save the sample list in ascending order i e from 1 to 4 145 Samples Experiment Tools Channels 1 2 Sample Statistic Cells live a Bjsrv2009 05 14_2053 027 100 0 7592 sv2008 12 10_2063 015 pos 100 0 14887 H v2008 12 10_2063 014 neg 100 0 18222 Ef v2008 12 10_2063 013 0ri 100 0 22130 C EAEE Ann n 14297 st f j Open Ctri O _st a E Add Import FCS file Export Sample Ungroup Resample 3 Click Apply to implement changes Click OK close the window To assign default settings for FCS Export 1 Click Edit Options Software and Export and Fcs Options Users Experiment E Instrument Software Version eile Fcs2 0 FCS3 0 Timers Acquire Export Format Statistic Best fit 16 bit Float FCS Backup H Regions Windows C Linear data Add ext info C Compatible Templates Views FCS Options Figure 6 49 Changing the default Fcs export settings 2 Use the check boxes and or radio buttons to activate deactivate a feature Version the default file export format can be either FCS2 0 or FCS3 0 Format Data can be saved as Best fit 16 Bit or Float The 16 bit format is compat
47. less than 3 m in length 1 5 Secure operation maintenance transport and disposal Observe the following instructions to ensure secure operation maintenance transport and disposal of your MACSQuant Analyzer IMPORTANT At all times local working area safety instructions laboratory policies and standards regarding laboratory health and safety and prevention of accidents must be adhered to 1 5 1 Secure operation If the instrument is not working properly and instructions or messages on the display screen advise to contact technical service secure operation is no longer possible Immediately switch off the MACSQuant Analyzer unplug the instrument from the electrical outlet and contact an authorized Miltenyi Biotec service provider or the Miltenyi Biotec Customer Support team 1 5 2 Servicing IMPORTANT Unless otherwise specifically noted in this User Manual or other Miltenyi Biotec documentation do not service the MACSQuant Analyzer yourself Servicing and repair must be performed by qualified service personnel Improper or incorrect servicing or repair of your MACSQuant Analyzer can cause hazards to users lead to unpredictable results device malfunction or damage premature wear and reduced life time of the instrument and may void your warranty Inquire with your local Miltenyi Biotec representative about Miltenyi Biotec s extensive instrument service and support arrangements or see www miltenyibiotec com support IMPORT
48. list oo CD45VioBlue A Y FITCA y CD19 PE A SPA A PE CY7A Y CD20 APC A V APCE A In a similar manner select deselect Features i e Channels and Functions i e statistics for export 4 After configuring the export options click OK 5 The file is exported to an Excel file and or the clipboard Note If the relevant Options check boxes have been selected use Windows explorer to navigate to the exported Excel file or alternatively paste the data into another windows application using the Edit Paste command 178 7 Shutdown of the MACSQuant Analyzer The chapter outlines how a manual and automatic shutdown procedure is made 7 1 Manual shutdown If no further measurements are required shut down the instrument as follows 1 Click to select data analysis mode or instrument off This automatically starts a washing protocol that lasts for approximately seven minutes which includes an incubation of the washing solution in the fluidics followed by the flushing of the washing solution and replacement with the storage solution Note Data can be analyzed using the software interface even after the instrument has been shutdown using the analysis mode 7 2 Automated shutdown An automatic shutdown procedure can be activated using the MACSQuantify Software This may be used to prevent the nonessential illumination of lasers which shortens diode lifespan or to guard against accidentally leaving the i
49. notices inform the user about potential risks if warnings and precautions outlined below are not followed The icon on the left side specifies the risk The hazard level at the top classifies the hazard as mentioned above The level type and source of the hazard as well as potential consequences prohibitions and measures are pointed as follows 1 1 2 Symbols The following chart is an illustrated glossary depicting the symbols that are used in this user manual and on the MACSQuant Analyzer Indicates a hazard situation which if not avoided could result in minor or moderate injury Indicates a hazardous situation which if not avoided could result in death or serious injury Attention consult the User Manual for further instructions and proceed with caution Warnings include the risk of damage to the equipment severe personal injury or loss of life Hazard of crushing and shearing Risk of crushing and shearing of bodily parts due to mechanical hazards Laser radiation Risk of serious eye and skin injuries Strong permanent magnet Contains a strong permanent magnet Magnetic devices can interfere with electronic devices or damage magnetic information carriers Risk of contamination if biohazardous material is used Indicates the risk of loss of life severe injury to the instrument operator or equipment damage due to potentially dangerous biological material Indicates the risk of loss of life or severe injury to th
50. of sample tubes They contain a coolant allowing racks to be pre cooled in the refrigerator for subsequent cooling of the cells during analysis The automated arm carries a needle port designed for computer controlled uptake of sample This needle is calibrated and self cleaned It is designed to move in the y and z direction Container for waste fluid The closure is equipped with a fluid sensor The closure the fluid sensor cable and the tubing connector are color coded red 209 Miltenyi Biotec Miltenyi Biotec GmbH Friedrich Ebert Strahe 68 51429 Bergisch Gladbach Germany Phone 49 2204 8306 0 Fax 49 2204 85197 macs miltenyibiotec de www miltenyibiotec com Miltenyi Biotec Inc 12740 Earhart Avenue Auburn CA 95602 USA Phone 800 FOR MACS 1 530 888 8871 Fax 1 530 888 8925 macs miltenyibiotec com Miltenyi Biotec Pty Ltd Australia Phone 61 02 8877 7400 macs miltenyibiotec com au Miltenyi Biotec B V Benelux macs miltenyibiotec nl Customer service Netherlands Phone 0800 4020120 Customer service Belgium Phone 0800 94016 Customer service Luxembourg Phone 800 24971 Miltenyi Biotec Shanghai Office Phone 86 21 6235 1005 macs miltenyibiotec com cn Miltenyi Biotec SAS France Phone 33 1 56 98 16 16 macs miltenyibiotec fr Miltenyi Biotec S r l Italy Phone 39 051 646 0411 macs miltenyibiotec it Miltenyi Biotec K K Japan Phone 81 3 56 46 8910 macs miltenyibiotec jp
51. overview of the entire gating strategy Note The regions in this gating strategy do not have the same hierarchy Regions are created within regions leading to subdivisions of gates regions 0 b MIN 2009 02 27 2053 021 F463 17691 1326 1328 Refer to section 6 13 2 for information on creating other gating strategies 12 To save the gating strategy as an Analysis template a Ensure that the MACSQuantify Software is in Analysis mode Al 7 b Click E anc Ji c Choose the file location Public Private or External d Name the file Click Save 164 6 13 2 Gating stratgies Note For background information about drawing regions gating with MACSQuantify Software refer to section 2 2 8 Classical hierarchical gating In section 6 13 1 an example of a hierarchical gating strategy is given In this classical Strategy gates are created within gates in order to identify sub populations of cells SMiN 2009 02 27 2053 005 100 0 eed aa4 OPPO reer irre rrrretrirrererirrretririiriiiiiiiiiiitiiiiitii iii Sc PA EZE 143622 E oh P2 60 9 139674 n C P3 20 4 46818 Regions having the same hierarchy It is also possible to create regions having the same hierarchy Regions drawn in a single plot have the same hierarchy In the example below regions P3 and P4 bottom left were both defined within Region P2 top right and therefore have the same hierarchy NOT gates So called NOT gates are us
52. powerful light emitting diodes LEDs for illuminating the reading area The 2D code 199 reader light is classified as a Class 1 laser product per standard IEC 60825 1 1993 A1 19976 A2 2001 maximum output 116 UW wavelengths 655 nm pulse duration 1 ms Please refer also to section 1 3 4 Important Information of the MACSQuant Analyzer Manual for associated warning and precautionary information The technical specifications of the MACSQuant Analyzer and the peripheral devices are as follows 12 2 Technical data and specifications of the MACSQuant Analyzer The MACSQuant Analyzer is labeled as a protection class device and must be plugged into a grounded power outlet see section 1 2 Warnings and precautions The main power supply cord and plug of the instrument shall comply with following specifications USA and Canada only UL listed and KAM cord minimum type SJ minimum 18 AWG 3 conductors Rated for a minimum temperature of 60 C Provided with grounding type NEMA 5 15P attachment plug rated 125 VAC 10 A Opposite end terminates in IEC 320 style connector rated 125 VAC 10 A Parameter Color Footprint Footprint with MiniSampler Height Weight Specification Green orange 605 mm x 400 mm w x d 605 mm x 500 mm w x d 392 5 520 mm adjustable touchscreen 50 kg Table 12 1 Dimensions of the MACSQuant Analyzer Parameter Input voltage Power consumption Specification Paramete
53. s Algorithms can be used to smooth the ie histogram Light medium or strong eu smoothing is available aen o amp Histograms can be display as a line chart or vi a bar chart The latter is used to a greater z degree 157 Region Regions The settings for regions and functions are functions identical to those for dot plots Overlay Population Several regions gated cell populations can be displayed on a single histogram In the following example four regions P1 P2 P3 and P4 were color coded and displayed Overlay _2063 015 pos P4 i Population 1 s1v2008 12 10_2063 015 pos P1 m Population 2 srv2008 12 10_2063 015 pos P1 P2 m Population 3 stv2008 12 10_2063 015 pos P1 P2 P3 m Population 4 sr 2008 1 2 10_2063 015 pos P3 P4 m 1 0e 21 0e 11 0e0 1 0e1 1 0e2 1 0e3 Table 6 9 An overview of the Properties settings for histograms Overview of the Properties settings for the Statistic option A Statistic text box is composed of a Header and a Table st 2008 12 10_2063 015 pos P1 P2 P3 P4 File am 2008 12 10_2063 015 pos mgd 2008 12 10 16 42 Slid pos 16 Descr T 0 YioBlue A Mean Yioblue A StdDeyr PE 4A Mean PE A StdDey a a f2 6o 20 97 arr 21 36 afore fade eu r0 af 20 83 Table Figure 6 63 A header and a table comprise a statistic text box A variety of display settings for the Statistic option can be modified u
54. the MACSQuant Analyzer the data is only copied to DVD Insert the backup DVD into the destination DVD drive of an independent personal computer on which the MACSQuantify Software is preinstalled This computer can be used for data analysis Start and login to MACSQuantify Software on the personal computer Click restore o d Note MACSQuant Analyzer data will be copied to the local drive of the personal computer After a successful data transfer the copied data will be marked as successfully copied on the DVD Note When performing a future data backup on the MACSQuant Analyzer ensure that this backup DVD is used 134 Performing subsequent MACSQuant Analyzer backup 9 Insert the designated MACSQuant Analyzer backup DVD 10 Click backup Note The MACSQuant Analyser software MACSQuantify will identify data marked on the DVD as successfully transferred to another computer This corresponding data will be deleted off the MACSQuant Analyzer hard drive and DVD before continuing with the backup procedure 11 After backup is finished remove the DVD from the drive 12 Transfer the data to an independent personal computer as described above g j Data written to DVD but NOT deleted P a from the MACSQuant 7 J Click wy Analyzer iia Ei i Backup Insert unused icon Backup is performed DVD RW data copied to DVD 9 N Previously backed up a Ea 2 marked dat
55. the container from the fluid container holder Do not disconnect the color coded tubing from the bottle closure 2 Place the new sealed container in its appropriate position Do not open a new fluid container until it is in position within the fluid container holder 3 Unscrew and remove the blue cap fasten the bottle closure securely 4 Remove the bottle closure while the container is still in the fluid container holder 5 Close the container with a cap and remove the bottle from the orange basket 6 Exchange the container with a bottle containing approximately 100ml of disinfectant e g 10 bleach 7 Attach the bottle closure 183 Note Be extremely cautious with the waste fluids and dispose of in accordance with institutional or governmental regulations 9 1 3 Exchanging the MACSQuant Column The MACSQuant Column should be exchanged every 3 months Exchange the column as outlined below Warning Latex gloves and protective eyewear should be worn at all times to protect against potential biohazard exposure 1 Open the front door and note the position of the tubing and MACSQuant Column in the MACS Cell Enrichment unit magnet Note The top end of the MACSQuant Column has a white filter outlined in the red box The column must only be inserted with the white filter at the top 2 Using both hands hold the top and bottom of the column and pull gently but firmly to remove it from its
56. to perform a system backup Visit www macsquant com to download the most recent version of the MACSQuantify Software from your personal computer or Apple Macintosh Copy the installation program to an empty memory stick i e ensure that no other files are already stored on the stick Note The memory stick MUST be labelled as MQSOFTSETUP If this is not the case please rename the memory stick using windows explorer desktop My Documents Y My Computer S Local Disk C E Desktop SSI Removable Disk D E E My Documents Expand nm mgef P My Conii Explore k on onnar E G Local Dik C Search Nn mgef a t rs z AutoPlay ver Milti D Sharing and Security tS ss Attach the memory stick to one of the USB ports located at the back of the MACSQuant Analyzer Login as administrator to the MACSQuant Analyzer Click Tools tab and Update Software Samples Experiment Tools Channels Show backup registry i Update software Import Export user settings MACS Quant live support Figure 4 3 Updating MACSQuantify Software on the MACSQuant Analyzer Follow the prompts to complete installation 72 4 2 1 Registering the MACSQuantify Software on the MACSQuant Analyzer Note If your copy of MACSQuantify Software is not registered please contact Miltenyi Biotec to obtain a registration code Visit www macsquant com for more information 1 Switc
57. two parameter dot plot showing side scatter plotted against forward scatter of human peripheral blood mononuclear cells PBMCs Three distinct cells populations can be identified according to their light scattering properties granulocytes monocytes and lymphocytes Scatter scales are usually plotted using a linear scale In fluorescence dot plots the x and y scales are used to plot fluorescence intensity and since fluorescence intensity can vary by several orders of magnitude between cells a logarithmic scale is usually employed e g fluorescence intensity spanning five decades for more information about choosing an appropriate scale refer the section Scaling flow cytometry data 24 below Dot plots are ideal for displaying relatively small numbers of events where discrete cell populations can be easily identified e g see Figure 2 1 They also provide some indication regarding the relative density of cell events i e with more events the dots accumulate forming a darker dot plot with more contrast However for a more accurate reflection of the relative density of cell events a density plot should be employed Histogram Histograms are used to plot the intensity of a single parameter x axis against the frequency of that parameter y axis i e the x axis represents scatter or fluorescence intensity and the y axis axis represents the number of events Since histogram charts can display only one dimension they should be emplo
58. using the MACSQuant Analyzer for the first time it is necessary to calibrate the hardware before calibration of the instrument settings This section discusses hardware calibration Note It is highly recommended that hardware calibration is only performed by an administrator who has been trained by Miltenyi Biotec Note In order to calibrate of the MACSQuant Analyzer the user must be familiar with the MACSQuantify Software Refer to section for an introduction to the MACSQuant Analyzer user interface 3 5 1 Calibration of the uptake unit 1 Click on the Tools tab and Calibrate uptake unit box File Edit View Mode Analysis Window Help admin mE Se Be SEBS a a eO EY Br Samples Experiment Tools Channels d Calibrate uptake uni Uptake unit calibration Calibrate rack detection 1 First push the Start calibration button to begin the calibration be careful the hardware moves Show backup registry 2 Move the needle and sampler by the hand to the specified position and press Save calibration 3 Test your calibration using the Test button select position you want to test Update software Import Export user settings Remove extemal media Time and date fo r Z offsst 0 S Start calibration calibration Save calibration _ calibration Test MACS Quant live support I p 9 10 1 __Stercaibra
59. with skin and eyes 186 4 Clean the washing station by wiping it with tissue and an appropriate disinfectant e g 70 ethanol isopropyl alcohol or 10 bleach Finally rinse with distilled water 9 1 8 Cleaning the uptake port 1 The uptake needle of the robotic arm should be cleaned regularly in order to prevent contamination or blockages Turn OFF the instrument 2 Wipe the needle with tissue soaked with 70 ethanol isopropyl alcohol or alcohol swabs followed by distilled or deionized water Move the needle holder up and down to get access to the entire surface of the needle 9 1 9 Decontaminating the MACSQuant Analyzer If the instrument has been used to process biohazardous samples it is recommended to decontaminate the fluidics system by running the standard shutdown sequence In case of spillage it is recommended to use the appropriate disinfectant for the potential pathogen e g MACS Bleach solution Use tissues or swabs to decontaminate surfaces The uptake needle of the robotic arm and the surface of the instrument also can be decontaminated upon contact with biohazardous samples Note Dispose of tissues and swabs appropriately It is recommended to wear protective gloves protective clothing and safety glasses to prevent contact with skin and eyes 187 10 Troubleshooting The instrument automatically analyzes the functionality of several hardware components during the instrument initialization procedure
60. 1 01 10 11 12 13 CD3 PE A CD14 APC A 1 02 1 01 10 11 iz 13 CD3 PE A Figure 5 18 Analysis mode CD14 cells isolated from PMBC using MACS MicroBeads were analyzed using the MACS Control MC CD14 Monocyte Cocktail human The positive fraction is shown A total of 1 56x105 viable monocytes were enriched from the original PBMC fraction which accounted for an enrichment rate of 69 7 The analysis template CD14 automatically generated the gating strategy and associated statistics 5 8 Reading reagents with the code reader in Express mode The 2D code reader barcode reader is used to scan reagent vials Reagent vials are automatically recognized and logged by the MACSQuantify Software To scan reagents perform 1 Click the activate code reader icon the following M The code reader will being blinking 2 Present the reagent vial in front of the 2D code reader Ensure the 2D code is facing the blinking code reader light The optimal reading distance is 0 5 2 5 89 cm from the code reader cover tilt the vial as depicted in Figure 5 19 Figure 5 19 Scanning a reagent using the MACSQuant Analyzer 2D code reader 3 Scanned reagents are reported by a MACSQuantify Software dialog box Note When scanning MACSQuant Calibration Beads the instrument will prompt to initiate the calibration procedure gt Calibration Particles detected LOT 5080414001 Would you like to proceed with calibratio
61. 1 0e1 1 0e2 1 0e3 l Oe De 1 Qe 2 1 0e1 1 0e0 CDOB FIT C A 1 0e CD6_FITC A Figure 3 38 Cells stained with CD8 FITC were optimally compensated right for FITC detection in the FL3 channel Note It is not possible to subjectively estimate what fraction of the signal needs to be subtracted compensation settings must be individually optimized for all activated fluorescence channels as well as for each fluorochrome and fluorochrome combination used An optimal compensation is of particular importance to distinguish dimly labeled cells from a negative population 3 7 1 Performing auto compensation Compensation should only be performed if the instrument has been successfully calibrated and when the MACSQuant Analyzer reports a MACSQuant ready Calibration OK status Automated instrument compensation is based on a 7x7 matrix It is 60 recommended that the MACSQuant Analyzer automatically calculates the appropriate compensation settings by using the automated compensation matrix and the compensation program matrix method However more advanced flow users may wish to adjust these settings manually using sliders classic method Using either single stained cells or compensation beads the MACSQuant Analyzer can accurately and reliably perform automated compensation based on a 7x7 matrix each channel compensated against the other 6 channels The utilized cell type and its respective surface marker should be brightly expressed on cells For ex
62. 1 512 1290 Fax 34 91 512 1291 macs miltenyibiotec es United Kingdom Miltenyi Biotec Ltd Almac House Church Lane Bisley Surrey GU24 9DR Phone 44 1483 799 800 Fax 44 1483 799 811 macs miltenyibiotec co uk 206 14 Limited warranty Except as stated in a specific warranty statement which may accompany your MACSQuant Analyzer the Product or unless otherwise agreed in writing by an authorized representative of Miltenyi Biotec Miltenyi Biotec s warranty if any with respect to this Product is subject to the terms and conditions of sale the Terms of the company within the Miltenyi Biotec group which supplied the Product The Terms may vary by country and region Copies of these Terms are available on request or at www miltenyibiotec com Nothing in this document should be construed as constituting an additional warranty Miltenyi Biotec s product warranty only covers Product issues caused by defects in material or workmanship encountered during ordinary use as described in the user manual or other documentation provided by Miltenyi Biotec it does not cover Product issues not arising out of defects in material or workmanship including but not limited to Product issues resulting from failure to follow installation operating and or maintenance instructions or environmental conditions prescribed in this user manual or other Product documentation misuse abuse neglect mishandling unauthorized or improperl
63. 29 A Gladbach Germany Figure 1 4 Warning signs for biohazard on rear panel and waste bottle Safety check After completing any service or repair work have your authorized Miltenyi Biotec service provider perform all safety checks required by the repair procedure to ensure that the instrument is in a proper operational condition 1 5 5 Transport The MACSQuant Analyzer should be transported with care in packaging specified by Miltenyi Biotec Internal damage can occur if it is subjected to excessive vibration or if it is dropped If the instrument needs to be shipped back to the manufacturer for service decontaminate the instrument from any hazardous material prior to shipment If you have questions regarding proper decontamination or shipment please contact technical service for assistance See section 9 1 9 for further information on instrument decontamination 1 5 6 Instrument disposal Please contact technical service for assistance if you wish to dispose of your instrument 1 5 7 Electromagnetic compatibility Changes or modifications of the equipment unless expressly approved by Miltenyi Biotec may void your authority to operate the equipment pursuant to 47 CFR 15 NOTE This equipment has been tested and found to comply with the limits for a Class B digital device pursuant to Part 15 of the FCC Rules These limits are designed to provide reasonable protection against harmful interference in a residential installation This eq
64. 6 9 4 Importing files MACSQuantify Software can import files in Flow Cytometry Standard FCS Formats MACSQuantify Software data and instrument settings can also be imported from an external file location for example a USB memory stick To import FCS files perform the following steps 1 Insert a memory stick or external media into the MACSQuant Analyzer USB port Alternatively ensure that the MACSQuantify Software has intranet access to the file location 131 d Import FCS file Look jn O dat 2 Click File an Figure 6 33 Importing an FCS File 3 Navigate to the file using windows tabs Highlight the file and click Open 4 The file will be imported To import MACSQuantify Software files from an external source perform the following steps 1 Insert the external media to the MACSQuant Analyzer USB port or computer USB port Note If no external device is attached to the MACSQuant Analyzer or personal computer the following error will be displayed External i No external media found 3 Select the file type Workspaces Instrument settings Experiments Reagents Analysis templates Data files 4 Click Open 5 The file s will be imported 6 9 5 Exporting files MACSQuantify Software data and instrument settings can be exported to a USB memory stick To export MACSQuantify Software files to an external source perform the following steps 1 Insert the memory stick to the MACSQuan
65. ANT When replacement or spare parts are required make sure that the service provider uses only genuine Miltenyi Biotec parts or third party parts specified and recommended by Miltenyi Biotec Using unauthorized replacement or spare parts can cause malfunction of the device and impair flow cytometry results Miltenyi Biotec does not honor any warranty or accept any responsibility for device failure or damages resulting from the use of inappropriate replacement or spare parts After completing any service or repair work have your authorized Miltenyi Biotec service provider perform all safety checks required by the repair procedure to ensure that the instrument is in proper operational condition Only use options and upgrades recommended by Miltenyi Biotec 1 5 3 External cleaning Unplug the MACSQuant Analyzer from the outlet before cleaning Do not use liquid or aerosol cleaning agents always use a damp cloth 1 5 4 Hazardous material Risk of loss of life if biohazardous material is used Wear protective gloves protective clothing and safety glasses to prevent contact with skin and eyes Operate device in a safety hood Decontaminate device after spilling of biohazardous material If biohazardous material is or has been used the operator shall choose and wear personal safety equipment in accordance with warnings and precautions for the used substances Wear protective gloves protective clothing and safety glasses to prevent contact
66. An error message will appear if action is required by the user Please see chapter 9 Maintenance for help with dealing with certain error messages otherwise contact the Technical Support 10 1 Problems not indicated by error messages This section addresses problems that are not indicated by a warning or error message that might occur during measurement Cell separation or rinsing programs Identify the problem and refer to the appropriate section 10 1 1 Column leakage 1 Ifa freshly installed MACSQuant Analyzer Column shows signs of leakage check if the column is installed properly The column should be inserted correctly into the column connector and fastened to the point of resistance If this is not the case loosen the column connector insert the column correctly and tighten the connector 2 Ifthe column leakage persists unscrew the column and check if the connectors of the columns are damaged If this is the case exchange the leaking column with a new MACSQuant Analyzer Column 3 If the problem still persists contact the Technical Service Note Check that the column is not broken or cracked In this event replace the column See section 9 1 3 for more details 10 1 2 Pump syringe leakage 1 Shut down the instrument 2 Remove the pump syringe as described in section 9 1 3 188 3 Carefully remove the plunger from the syringe Wet a tissue with distilled water and clean both the plunger and the syringe R
67. An example of single color compensation PE fluorescence measured in the FITC channel In the following example human peripheral blood cells were stained with R Phycoerythrin PE conjugated and Fluorescein conjugated FITC monoclonal antibodies PE and FITC and are both excited by the MACSQuant Analyzer blue laser operating at the wavelength 488 nm The emission spectra from each fluorochrome are detected by two discrete photomultiplier tubes PMT FITC by CH2 and PE by CH 64 3 Nevertheless there is unavoidable cross over of FITC fluorescence FL2 in the PE recording channel CH 3 and simultaneous cross over of PE fluorescence FL3 in the FITC recording channel CH 2 To correct for this so called fluorescence bleed instrument compensation is necessary see Figure 3 43 for illustration FITC PE bandpass FITC emission bandpass filter PE emission l spectra ii filter spectra gt gt a gt Normalized excitation emission Wi Bleed of FITC fluorescence 0 3 i into PE PMT Bleed of PE gt 0 2 gt fluorescence into FITC PMT WN N 0 1 0 EZ 2 450 500 550 600 650 Wavelength nm Figure 3 43 Excitation dashed lines and emission spectra continuous lines of Fluorescein FITC and R Phycoerythrin PE The wavelengths of the corresponding band pass filters used to restrict PE and FITC fluorescence to the respective FL2 and FL3 channels are also shown In spite of the us
68. Analyzer Washing Solution was developed for an optimal cleaning of the MACSQuant Analyzer tubing system and is provided in 1 5 L container MACSQuant Analyzer Storage Solution The MACSQuant Analyzer Storage Solution is a sterile filtered ready to use solution used for the overnight or long term storage of the MACSQuant Analyzer The MACSQuant Analyzer Storage Solution is supplied in a 1 5 L container and prevents the corrosion or contamination of the fluidics system during long or short term storage of the instrument Exchange of solutions within the fluidics system to the storage solution is performed automatically when the system shutdown protocol is activated in the software 3 3 2 Hardware and disposables The single tube holder has been designed for compatibility with all standard flow cytometry tubes 1 5 mL 2 mL and 5 mL tubes Note Should the MACS MiniSampler be used the three tube racks available Chill 5 Chill 15 and Chill 50 are designed to hold standard 5 mL 15 mL and 50 mL tubes respectively see Table 3 4 Additionally the Chill 96 rack and the 96 rack can be used for the use of 96 well microtiter plates Rack type Maximum number Order numbers of samples Chill 5 5 mL 24 130 092 951 Chill 15 15 mL 15 130 092 952 Chill 50 50 mL 6 130 092 953 Chill 96 96 rack 96 well microtiter 96 130 094 459 plate Reagent rack Reagent vials 130 094 574 Table 3 4 Rack types for the MACS MiniSampler and compatible sample tu
69. Click Apply to apply changes 2 Click Ok to apply changes and close the Properties window Overview of the Properties settings for Dot plots and Density plots A variety of display settings for dot plots histograms and density plots can be modified using the Properties option from the dropdown menu 1 Click the icon located adjacent to the dot plot histogram or density plot 2 Click Properties HAMM The properties window will appear Note Refer to Table 6 8 below for an overview of the various display Properties that may be modified for dot plots and density plots using the MACSQuantify Software 155 Tab Category Property Associated screenshot option All Data Fixed number Om O Percentile 50 v Fixed number 100g vial lt gt v 1000 vja lt gt Regions ie Region functions functions lt srv2008 12 10_2063 013 ori ame srv2008 12 10_2063 013 ori srv2008 1 2 10_2063 013 ori P s1v2008 12 10_2063 013 oni P r Coun stv2008 12 10_2063 013 o0ri P j l Description All events are displayed on a dot plot or density plot 50 25 5 2 or 1 percentile values of the total events can be displayed on a dot plot or density plot This is useful when too many events have been acquired and the displayed plot histogram is saturated A fixed number of events can be displayed on a dot plot or density plot Num
70. Click on the desired tab for example the Experiment tab or Data file tab to open an experiment definition or data files respectively Public sa Private External Directory Data files sis data _sr 2009 02 04_2062 054 pos PBMC Analysis E PBMC stv2009 02 04_2062 052 ori E MC_CD4_T_Cell_Cocktail_h st 2009 02 04_2062 053 neg E MC_CD3_Pan_T_Cell_Cocktail_h stv2009 02 04_2062 054 pos me MC_CD1S_B_Cell L E MC_CD14_Monocyte_Cocktail_h iLa17 E CD34cocktail hlog E CD34_CD133 cocktail log5 E CD34_CD133 cocktail hlog E CD34 Stemcell log5 E 2009 09 01_Calibration E 2009 04 28 CD34 Stem Cell Analysis Figure 6 30 Highlight the desired tab to view available files The Experiment left or Data files right tabs were selected in this example Note Multiple data file types can be selected and opened at once right To open workspaces 1 Highlight the Workspace tab on the Open window 2 Highlight the file location External to open files from external media e g a USB memory stick Files are opened from the default Private location 3 Select the file type and click Open L on To open Instrument settings 1 Highlight the Instrument tab on the Open window 2 Highlight the file location Private Public or External 129 3 Select the file type and click Open L To open experiment definitions 1 Highlight the Experiment tab on the Open window 2 Hig
71. D Pin 1 RED Pin 2 GREEN Pin 3 BLUE Pin 5 GND Pin 6 GND Pin 7 GND Pin 8 GND Pin 10 GND Pin 13 HSYNC Pin 14 VSYNC Table 12 2 Power and interface port technical specifications of the MACSQuant Analyzer Conditions of operation 201 Conditions of operation 20 25 C with 0 85 humidity at a maximum altitude of 2 000 m Supply voltage fluctuations up to 10 of the nominal voltage Transient over voltages voltage in excess of the normal operating voltage present on the mains supply Category Il The instrument is suitable for rated pollution degree 2 The MACSQuant Analyzer is not specified for use in a cold room The MACSQuant Analyzer has been investigated by Underwriter Laboratories in accordance with the standarads UL 61010 1 CAN CSA C22 2 No 61010 1 and IEC 61010 2 081 and meets the intent of the directive 2004 108 EC electromagnetic compatibility and 2006 95 EC low voltage equipment Compliance was demonstrated by conformance to the following harmonized European Standards which have been listed in the Official Journal of the European Communities EMC EN 61326 1 cus C amp EN 61000 3 2 EN 61000 3 3 Low voltage equipment EN 60825 1 EN 61010 1 EN 61010 2 081 Compliance was demonstrated by conformance to the following Codes of Federal Regulations 47 CFR 15 class B CDRH 21 CFR 1040 10 and 1040 11 12 3 Technical data and specifications of the MACS MiniSampler Model MACS MiniSampler
72. EDs Invisible rack detection lasers VCSELs are located within the rectangle area The 2D code reader visible is located within the open circle 1 4 Secure installation This section describes the requirements your site must meet for safe installation and operation of your MACSQuant Analyzer Read the instructions in this section and ensure that your site is properly prepared before you connect the instrument to its power source When planning your site layout and equipment locations keep in mind the precautions described in this section to help avoid instrument failures and reduce the possibility of environmentally caused shutdowns IMPORTANT At all times local working area safety instructions laboratory policies and standards regarding laboratory health and safety and prevention of accidents must be adhered to 1 4 1 Mounting accessories Do not place the MACSQuant Analyzer on an unstable table cart stand tripod or bracket As a consequence the instrument might fall down This may cause serious bodily harm and or serious damage to the instrument Use only on a table cart stand tripod or bracket that can easily support a weight of 50 kg Do not place the MACSQuant Analyzer within a built in apparatus or a confined space such as a shelf rack unless the apparatus has been specifically designed to accommodate the instrument proper ventilation is provided and the mounting instructions for the instrument have been followed 1 4
73. ETS OOOO0OO0O i Figure 5 5 Location of the Sample ID and Description fields 80 Entering Sample ID and Description text 1 Click on the Sample ID or Description text box 2 Either the appropriate alphanumeric text using the keyboard 3 The data is automatically stored in the field 5 5 3 Mode The Mode dropdown box has two options e Analysis template Analysis templates simplify data analysis so that even inexperienced flow cytometry users can perform complex data analysis Analysis templates e g gating strategies can be only created by administrators or custom users For example the following gating strategies were defined by an administrator or customer Cell Cycle odora cells Cell Cycle Analysis e Analysis The Analysis option from the Mode dropdown list reveals a list of options available to perform flow cytometric cell analysis For example CD14 allows for analysis of cells separated using CD14 MicroBeads i e application of the MACS Control CD14 Monocyte Cocktail gt e a4 wm amp NS Help Custom ont Definition Rack m Semple ID Description 7 C a Figure 5 6 Location of the Mode dropdown box Note Analysis templates or Analysis options cannot be created or modified by Express users Selecting an analysis template mode 1 Select Analysis template from the Mode dropdown menu M ETHGE 81 2 From t
74. I PE Cy5 1 2 2 A amp 36 oo so gt Figure 6 20 Chill 5 tube rack was selected for multisample labeling 123 2 Left click once on rack coordinates Al and A2 oO 7 2 3 4 000000 ooooo0o0 E C C Ae a E ae eee eo F i a a ry e D a J as P a Clear Group Figure 6 21 Measure and select The settings for sample positions Al and A2 may be modified e g a labeling strategy may be applied 3 Define the experiment settings for positions Al and A2 as shown below see section 6 3 6 for more details on defining experiments Samples Experiment Tools Channels Experiment Rack Chill 5 tube rack O File Triall 001 vja O Project MACS Control Cocktail CD14 d Sample ID Samples 1 amp 2 Description Human PBMC Flow rate Annotations Autolabel Settings Hi l bow Med igh FLI CD45VioBlue FL5 PE Cy7 Pickup and measure g Mix sample FL2 ale FLE CD15APC FL3 CD14 PE FL APC Cy Mode Standard v Uptake volume 50 p LJ Sample volume 200 pli fy i Settin 7 DoF ENRerErESr yee Autolabel Settings Annotations Autolabel Settings Custom Express C lt add gt Type Analysis M C lt add gt Mode MC_CD14_h v C lt add gt C lt add gt C lt add gt a Time Titer t 10 la 1 10 via 5 via i Wea 32 ca a Ft 6 Ca ae Catal aol a 0 O Jel Eaz e 0
75. IT plion Lew ard ipid se i une i BZ colors compensation Z 4 2 rel vaime n 2 TEER rere Create your region of Interest and il tat press continue to proceed 1 o A 182 183 101 181 162 103 Continue APC A live 133 i rA 4 2 KE S ia 8 8 X 1 101 te 4 1 a S 10 le je2 e3 401 w w2 m PI PECJ5A APCA a 0 0 Figure 3 47 7 color compensation is underway The user is prompted to select a region of interest before continuing 68 Note Selecting a region of interest is highly recommended Depending on the experiment a particular cell type or population may be selected for compensation 14 The MACSQuant Analyzer will perform 7 color compensation on the selected region of interest Fle Eck View Mode Analysts Window Help admin gt PCHCREEER GENUA CEART Sample Expeimsh Tock Crevice Epaima Rack HIE nber Fie amanan a Proje SampleID Vistas Figure 3 48 7 color compensation is underway for Region P1 15 Following completion of compensation the user is prompted to save the data into the current compensation bank Click Yes to save current settings Click No to abort the process FRB d mew vece Anayas Window Hep admin Hga a Peo fenfof ese all fa val Ohl S bubs peck adrcl09 3 00 HR Figure 3 49 7 color compensation was successfully completed 69 4 Installation of the MACSQuantify Software This chapte
76. Miltenyi Biotec MACSQuant Analyzer amp MACSQuantify Software User manual Version 2 MACSQuant Analyzer amp MACSQuantify Software User manual Version 2 0 Miltenyi Biotec GmbH Friedrich Ebert Strake 68 51429 Bergisch Gladbach Germany Phone 49 2204 8306 0 Fax 49 2204 85197 E mail macs miltenyibiotec de www miltenyibiotec com No part of this user manual may be reproduced stored in a retrieval system transmitted published or distributed in any form or by any means electronically mechanically by photocopying microfilming recording or otherwise without the prior written consent of Miltenyi Biotec however notwithstanding the foregoing the owners of the MACSQuant Analyzer may make copies solely for purposes of training personnel in the use and servicing of the unit within their business or organization Maximal care has been taken by Miltenyi Biotec in the preparation of this user manual However Miltenyi Biotec shall not be liable for any technical or editorial errors or omissions contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this document The information in this document is provided as is without warranty of any kind and is subject to change without notice MACSQuant MACS and the MACS logo are either registered trademarks or trademarks of Miltenyi Biotec GmbH or its affiliates in Germany the United States and or o
77. NSTALLA TION abwuucteiuecostvucdvenserevatwnveeetnautouundebsunchnavedeaatt E a aaan 14 tA I MOUNTHAG ACCOSS OOS sorse r E E E a a 14 FAZ AIP GH CULATION eaetiilete EA E E a E E 14 1243 Water ANG MONS TUT E erior E E E EEE TEE T E 15 1 4 4 Grounded earthed Produit wiacccccenccuenccuenseuensenensnnensnnenunnensensnnenunneuaneguunsenensnsenes 15 14D T OWON SOUCO S orib EE AEAEE tas EEEE S 15 FAO AACCOSSIDIMCY cvs towel EEE E NE EA E EE NE EREE 15 FALL PTIDMCTAl COVICES ossia ae a ie diate N EE E Ni 15 1 5 SECURE OPERATION MAINTENANCE TRANSPORT AND DISPOSAL 0cececececeeeeeeteveeeeeeeneneevanaeeeenaes 16 ha TSEC EC ODO AON aleschiasaiaiedint tes cdudshaletuba neue idaledbotuevidecisneiidabessoaedousien duiuevaobascdetee 16 iD ae CR VIGIING EEEREN O uel acacia uate rdaneenel staat baste catedoe AATA 16 FSS EX TETAN ClCAINAG oeren EENET A E a AE I7 1354 Hazardous Malena lkiie eie A E EEEE EEE E EREET EaR I7 hI I ANS OOM oiei iE ESEESE RANE E EEEE A EEES 18 ka bAs tument di POSA eikean eia E TETE ATA eet 18 1 5 7 Electromagnetic COLD ATIDINITY cacccencneccuncneccunensesecenscsetensnsenensnsaueteneasenensasesensnensanes 18 2 Modu TION aida sett cits ana aeons cnc re ceases eae gaated tama der eaeaea ewe esonatetarcaees 20 ZAM PURPOSE se cenaatcctncasnoet A sich cnatestetdca date sande E a teeeranes 20 27 PAPPIIGATION Susan eaden tenting e a tee atcmee eaten cneneee te eacact aes enews eat icena en tse te tana een eaee 2 221 FIVOLCSCCNCE CCl ANA
78. ST L Annotations Autolabel Settings FLI FL5 na me as e BOOOO0O0 0 FL3 FLI FLA Group A ie ar ae Na Res sa is er as I EA B Bla toe aes DN oN on baw sa ed or No E aa a cata raa ao a a a a ey eee ieee NA Oe fo CN wow hoe AN Sat aot Os N D ia aa fc Ned N A AA N Figure 6 17 The Chill 5 tube rack template was displayed Note The popup rack window can be closed or opened at any time using by clicking the Rack icon Note This information is also relevant to section 6 3 6 Define an experiment in Custom mode of this manual Sample racks are represented graphically by the MACSQuantify Software All rack positions are given by coordinates columns are assigned numbers rows are assigned letters To select a single rack position use the left mouse button single click to activate the desired rack coordinate alternatively the MACSQuant Analyzer touchscreen may be used 119 Figure 6 18 Configuring a sample rack using the MACSQuant Analyzer touchscreen e When a rack position is selected for the first time by a single mouse click the following Status is reported e By re selecting the same rack position the status changes from to J An explanation of the various rack configurations for single sample positions is given by Table 6 6 User action with left mouse Effect Details button or fingertip action on touch screen De
79. Size without lid 182 mm x 148 mm x 47 mm Size with lid 280 mm x 153 mm x 172 mm Weight 1 5 kg Input voltage 24 VDC Current 0 8 A Sub D9 interface Pin 1 NC with shielding Pin 2 CAN Pin 3 GND Pin 4 NC 202 Pin 5 24 V Pin 6 GND in 7 CAN Pin 8 NC Pin 9 24 V Table 12 3 Technical data and pin assignment for MACS MiniSampler Note The MACS MiniSampler is labeled as a protection class IIl device and must only be plugged into the connector labeled with external CAN on the MACSQuant Analyzer For further details see section 1 2 Warnings and precautions Note The MiniSampler unit is an optional extra If the MiniSampler is not ordered a dongle will be shipped with the MACSQuant Analyzer It is essential to attach the dongle to the external MiniSampler CAN socket located at the back of the MACSQuant Analyzer The MACS MiniSampler is labeled as a protection class Ill device and must only be plugged into the connector labelled with External CAN of the MACSQuant Analyzer see chapter warnings and precautions The MACS MiniSampler is designed for operation with three different tube racks and a reagent rack Rack type Slots Chill 5 24x5 mL Chill 15 15x15 mL 5x5 mL Chill 50 Chill 96 96 well rack 96 microtiter plate rack Table 12 4 MACS Cooling Tube Racks Chill Racks 5 15 and 50 Rack type Slots MACS Reagent Rack 4 4 x MACS Reagent vials Table 12 5 MACS Reagent Rack 4 Co
80. VD as successfully transferred to another computer This corresponding data will be deleted off the MACSQuant Analyzer hard drive and DVD before continuing with the backup procedure 93 11 After backup is finished remove the DVD from the drive 12 Transfer the data to an independent personal computer as described above Data written to DVD but NOT deleted from the 4 kik Insert unused icon ra Backup is performed DVD RW data copied to DVD a N Previously backed up marked data are Insert DVD into personal i MACSQuant f Click O d Analyzer Gi i Backup Click 7 deleted from the DVD an puter with Backup o pus MACSQuant MACSQuantify nalyzer 3 Software J Click Restore A meen maed ay tae data ae dealer the icon ata backup as successfully backed up copied to the DVD RW Y computer Figure 5 21 Schematic of the MACSQuantify Software MACSQuant Analyzer DVD backup procedure 5 10 2 To perform backup to a USB memory stick 1 Ensure that no network drive has been defined as the default location for backup files Please contact your administrator for further advice 2 Insert a memory stick into the MACSQuant Analyzer USB port or USB port a personal computer Wait a few seconds 3 et 4 The files will be automatically written to the USB memory stick Restoring files from a USB memory stick to a personal computer 5 Start MACSQuantify Software an
81. a are 5 eae pee ie into Click i deleted from the DVD a et Sait Backup 2 and MACSQuant nae B comp i P Analyzer o he MACSQuantify A Software _ _ i J i i Ves Click Restore mer maed ye data an italia the icon data backup as successfully backed up copied to the DVD RW computer Figure 6 34 Schematic of the MACSQuantify Software MACSQuant Analyzer DVD backup procedure 6 10 2 To perform backup to a USB memory stick 1 Ensure that no network drive has been defined as the default location for backup files Please contact your administrator for further advice 2 Insert a memory stick into the MACSQuant Analyzer USB port or USB port a personal computer Wait a few seconds 135 3 Click O 4 The files will be automatically written to the USB memory stick Restoring files from a USB memory stick to a personal computer 5 Start MACSQuantify Software and login to a user account 6 Insert the memory stick into a USB port of a personal computer with MACSQuantify Software installed 7 Click File and Import External media More than one external device found Please select one and press ok se REMOVABLE D vi REMOVABLE D swe REMOVABLE E Figure 6 35 If more than one external USB is attached you will be prompted to selected the correct external device 8 Use the dialog box to select the type of file for import e g Workspace Instrument settings Data files etc
82. a e Iz Figure 3 26 Chill 5 tube rack is successfully calibrated Calibration of MACS Chill 96 well plate optional step 1 Ensure that the MACS MiniSampler is correctly attached to the instrument This includes fastening of the corresponding cable to the External CAN port located at the back of the instrument see section 3 2 3 for information about correct connection of the MACS MiniSampler 5 2 Click the Experiment tab and select the 96 well format from the Rack pull down menu Samples Experiment Tools Channels Experiment Pak E O File adm2003 09 07 001 fa O Figure 3 27 Selecting a 96 well rack for rack calibration ensure the checkbox is activated 3 Place an empty 96 well plate on one of the 96 racks and place both onto the MACS MiniSampler 4 Click Start calibration The needle arm will automatically insert the needle into a rack position H12 specified by the MACSQuantify Software ples J Calibrate uptak Zoffset 0 gt Start calibration Save calibration Test Home pos 0 00 mm Single tube rack Start calib Offset 64 90 51 10 3 30 mm Chill 5 tube rack Zoffset 0 gt Start calibration Save calibration Test Offset 13 60 44 70 0 10 mm 96 well Zoffset 0 Start calibration Save calibration Test O 123456789012 Offset 13 90 45 30 4 00 mm Reagent Rack 4 Offset 11 60 45 40 4 00 mm B c D
83. ack and Chill Rack 5 The MACS MiniSampler Figure 12 5 an optional attachment is designed for automated multiple sample processing by the MACSQuant Analyzer The MACS MiniSampler can be loaded with MACS Chill Racks that hold cell samples and cell separation fractions The upper plate of the Mini Sampler moves horizontally x plane of direction and aligns the tube openings with the port of the automated arm The guide of the MiniSampler is directly attached to the corresponding slot located below the washing station The MiniSampler is automatically detected upon attaching the MiniSampler sensor cable to the corresponding socket at the rear of the instrument The type of tube rack carried by the MiniSampler can be automatically recognized by the rack detector using the rack detection barcode reader Figure 12 6 or the user can specify which rack will be used During operation the tube rack should be covered with the Mini Sampler lid that is connected to the lid guide The MiniSampler can be disconnected from the MACSQuant Analyzer by gently lifting the MiniSampler in an upward direction followed by carefully pulling the device away from the MiniSampler slot located on the MACSQuant Analyzer 2D code reader Code reader Rack detector MACS MiniSampler a a F Figure 12 6 Expanded view of the MACSQuant Analyzer 2D code reader barcode reader The MACSQuant Analyzer is equipped with a 2D code reader that uses lasers and
84. ad cells and debris B A region P2 was defined within gate P1 to select for viable CD34 cells Any remaining dead cells are positive for Pl and are excluded from the region P1 P2 C The region P3 was defined within gate P1 P2 to select for all viable CD34 cells Region P3 was renamed CD34 target cells for added clarity D The final gate is displayed namely P1 P2 CD34 target cells The corresponding statistics are shown by the adjacent table E To demonstrate the gating strategy all defined regions were color coded are display on a single two parameter dot plot P1 is green P1 P2 is red P1 P2 CD34 target cells is blue The black dot plot events were excluded by region P1 For a more detailed explanation on this gating strategy download the product data sheet MACS Control MC CD34 Stem Cell Cocktail human order number 130 093 427 2 3 MACS Cell Separation MACS Cell Separation i e the magnetic separation of defined cell populations using MACS Technology is widely regarded as the gold standard in cell separation MACS 29 Technology is based on the use of MACS MicroBeads MACS Columns and MACS Separators strong permanent magnets MACS Technology can be used for the targeted cell enrichment or depletion of cell types or populations through their expression of particular surface antigens MACS Technology provides the means for the pre enrichment of rare cells for subsequent flow cytometry analysis In
85. aken while handling fluids Clean up spillages immediately Do not allow fluids to enter the interior of the device Unplug the power cord before manually cleaning the MACSQuant Analyzer Warning A potential risk exists if an opened dropped or damaged MACSQuant Analyzer is used if liquids are spilled into the instrument if an object has entered the instrument through the ventilation slots or if an object has been dropped into the instrument If flames or smoke appear immediately switch off the MACSQuant Analyzer unplug the instrument from the electrical outlet and contact an authorized Miltenyi Biotec service provider or the Miltenyi Biotec Customer Support team Use of a damaged instrument or an instrument with a damaged power cable is expressly prohibited 1 3 2 Strong magnetic field Extremely strong magnetic fields Risk of severe personal injury to persons carrying pacemakers or electronical medical implants stay clear Keep all magnetically sensitive objects away from device Warning The MACSQuant Analyzer is equipped with an extremely powerful magnet Keep any magnetic information carriers such as credit cards magnetic tapes and floppy disks and any electronic equipment such as hearing aids pacemakers measuring and control instruments computers and watches at a distance of at least 20 cm from the magnet cover These items may be affected or damaged by the magnetic field Figure 1 1 Location of warning s
86. alog box After a few seconds the main screen will appear in Data analysis mode 6 3 3 Activate the touchscreen keyboard on the MACSQuant Analyzer The touchscreen keyboard can be used to enter information into the Sample ID and Description fields Users may find it easier to use a conventional keyboard and mouse which may be connected to the back of the analyzer as described in section 12 1 To activate the touchscreen keyboard perform the following 1 Click the Keyboard icon 2 The Keyboard popup window will appear 3 Click the Keyboard icon once again to close touchscreen keyboard 6 3 4 Check the fluid levels Check that sufficient running buffer and washing solution are present in the containers for the measurement minimum 150 mL of each Replace any of the solutions whose levels are low Also check that the waste container is empty Please note the bottles are only illuminated after the system has been placed into acquisition mode If any buffers or solutions need to be changed 1 Press the Main Instrument Control icon to set the instrument into Acquisition mode This will initiate the priming of all of the fluidics turn on the illumination LEDs for the four buffer bottle and turn on the lasers Each time you select the Main Instrument Control a dialog box will appear providing two of three options 104 2 3 4 5 Analysis mode Acquisition mode or Instrument off Fie Edit view Mode Analysis W
87. ample control sample aliquots of a peripheral blood mononuclear cell PBMC preparation are often singly stained using CD8 FITC CD8 PE or CD8 APC antibodies in order to compensate fluorescence signals in FL2 FL3 and FL6 Using individually stained live cells for compensation 1 Label the cells individually using appropriate fluorochromes that correspond to the fluorescence channels of interest Immediately prior to measurement transfer equal volumes of all single stained cell aliquots including a negative sample into one tube and place in front of the MACSQuant Analyzer For further information concerning available reagents and corresponding Staining protocols please refer to the Miltenyi Biotec catalog and or datasheets at www miltenyibiotec com and www macsquant com 2 Select the fluorescence channels that require automatic compensation From the menu bar select Edit and Calibration or Ctri Alt C to open the Calibration settings dialog box Select which channels require compensation using the Comp drop down list based on the following nomenclature Comp Yes fluorochrome particle P in this channel Comp Yes __ no fluorochrome particle in this channel but the amount of fluorescence spillover into this channel needs to be determined Comp No__ no fluorochrome particle in this channel and the compensation value in this channel will not be determined 61 Calibration settings Banks De
88. ase click OK File Edt View Mode Analysis Window Help admin x Xie gt Ally E Help Logout 0 Samples Experiment Tools Channels T Calibrate uptake unit Calibrate rack detecti Uptake unit calibration 1 First push the Start calibration button to begin the calibration be careful the hardware moves 2 Move the needle and sampler by the hand to the specified position and press e calibration 3 Test your calibration using the Test button select position you want to tesi Needle arm calibration relative to washing station Zoffset 0 gt Start calibration Save calibration Test Home pos 0 00 mm Measure pos 29 10 44 50 mm Reagent Rack 4 calibration A Be sure the Reagent Rack 4 rack is plugged in and single tube rack is disconnected pon test 4 30 mm Zoffset 0 Start calibration F Save calibration Test Offset 13 60 44 70 0 10 mm Zoffset 0 gt i Start calibration Save calibration Test Offset 13 90 45 30 4 00 mm Reagent Rack 4 Z olfset 0 Start calibration Save calibration Test Offset 11 80 45 50 4 10 mm The needle arm will automatically be positioned in reagent vial position Al the position is correct click Save calibration Click Test to test the calibration The needle arm should be correctly positioned in all four vials File Edit View Mode Analysis Window Help admin x x iS AGT E Help Logout Q a SS Se SSS SSS SS SS
89. ast to remove dead cells and debris from a dataset This can be achieved by defining regions of interest or gates around certain cell populations These gates are defined using geometric shapes and can be included or excluded in subsequent data analyses The following geometric shapes can be used by the MACSQuantify Software to define regions MACSQuantify NETS DECUS s KLaauug Uf 16 U01 Ellipse oe Rectangle te 0e 1 020 1 07 1 Ce De 11N3 PF A PE A I PA a 7 76 PE Software icon a Polygon Freehand shapes can be drawn i Quadrant Two parameter dot plots can be subdivided into four quadrants UL Upper left LL Lower left LR Lower right UR Upper right Intervals or markers can be drawn on ata histograms to calculate statistics for 7 designated regions pream Table 2 2 Geometric shapes that can be used to draw gates or regions of interest using the MACSQuantify Software Complex or boolean gating Several regions can be defined to form a boolean argument or a gating strategy which only displays highly specific cell populations for example cell populations with a defined set of scatter properties and or a specific cell surface marker phenotype as 28 identified by fluorescence labeling using fluorochrome conjugated monoclonal antibodies Gates or regions created using MACSQuantify Software are identified by the letter P where P is derived from Popul
90. ata It is possible to recompensate data that was already acquired This is of benefit if data was acquired using incorrect instrument settings The results can be reanalyzed using a different compensation matrix Note Refer to section 3 7 for an introduction to compensation and explanation of how instrument compensation is performed To recompensate saved data 1 Open the desired data file s The files will be loaded and can be view from the Sample tab 2 Click on the Sample tab menu 3 Apply instrument settings analysis template as instructed above see Applying previously saved instrument settings PMT voltage and compensation settings 4 Click a to view the instrument settings Experienced users can modify the Channel and Compensation values if required Click Apply to implement changes to the compensation matrix 171 5 Right click on the data file and select Recompensate gt open R E Add Import FCS file f Export Sample Apply instrument settings Apply analysis template View with Analysis cd1 Export sample list Figure 6 74 Recompensating a data file It is also possible to recompensate all data files that have been opened and are shown in the Samples window Note To recompensate all opened data files select Recompensate all Note Recompensated files are prefixed with an underscore i e _srv2009 05 14_2063 015 has been recompensated Samples Experim
91. ated CAIIDIACION ic cceccccccenenenenenenenenerenenenesesesesesesnenenenenens 56 3 6 2 Performing manual calibration vicacccececucenenenecensecseuenesenenensnsaeaeesusesereneneneaeasasesenens 59 3 7 COMPENSATION OF THE INSTRUMENT SETTINGS cccccecceceeveceevaceeeateaeaeeaueneaueavenvavenvausevaeeeeanses 60 3 7 1 Performing AUTO COITIPCISACION as ccccceccccecenenenensnsnenenenenenenesesesesesesesesnssesenenanenenans 60 3A PF COIOF COMPENSA ON orere neer dusiasiaae at alnd boiulbe Seu ea ENEE RERA 66 4 Installation of the MACSQuantify Software ccccccceseeeeeseeeevaveeeeeeuavanseeneneanans 70 4 1 INSTALLING SOFTWARE ONTO A PERSONAL COMPUTER csecceeeceeeeeaeeevaceevaceeuaeeeeaneauenvanenvansuvanes 70 4 1 1 Registering the MACSQuantify Software ON a personal COMMPUCES crscececenenenenseees Zi 4 2 UPDATING THE MACSQUANTIFY SOFTWARE ON THE MACSQUANT ANALYZER ccceeeeeeeeeeeeeueens 72 4 2 1 Registering the MACSQuantify Software on the MACSQuant Analyzef 73 5 EXpress MOGE sessiiss tee man a Waae cincanse 74 5 1 QUICK GUIDE TO THE EXPRESS MODE MAIN WORKSPACE ccecceceeeaceeeaceaveuvaceeuaceeuaeeneaneaeeneatentass 74 J2 LOGIN TO EXPRESS MODE serierne a E a 76 5 3 SWITCHING TO EXPRESS MODE FROM CUSTOM MODE 2ceeceeceeteceeteateseeatecteaeecteeeateateateaeeteaees 76 5 4 USING THE TOUCHSCREEN KEYBOARD ON THE MACSQUANT ANALYZER 00ceeeeceeeeeeeteeeceateatentaes 77 5 9 DEFINING ANP EXPERIMENT oreen a E a 77
92. ation An example of a complex gating strategy is shown by Figure 2 6 Sensitive rare cell analysis of CD34 cells was performed using the MACSQuant Analyzer In order to visualize pre enriched human CD34 cells froma peripheral blood mononuclear cell PBMC preparation a suitable gating strategy was devised Figure 2 6 A to E Cells were autolabeled with PE conjugated anti human CD34 antibodies to detect for CD34 cells Propidium iodide solution was used to exclude dead cells from flow cytometric analysis An explanation of the gating strategy is given in the accompanying figure legend 2003 01 30_2062 008 1 30_2062 008 4P1 _ FOE 1 0e3 1 0e2 lt Lia k 1 0e1 lt Gate P37 has i a a F SE 1 ded En been named ek CD34 target cells Data ae ae et ee excluded 1 oe2 a from analysis 4 250 s500 750 1000 1 0e 21 0e 11 0e0 1 0e1 1 02 1 023 F5 C A PE A 008 CD34 target ce eden 2008 111 30_2062 008 t Tl Fie ode a aieia aradi 13 28 K Sid MC COJA par Dert 750 FRiegion x X T Count A ee cot Ca ae a adii2008 01 30 _2062 008 100 00 100 00 as G cag e aa A O PI aL 770 7270 a _ T a i 2 at mene oe q in F2 7265 5355 aa E CDH tegat calls a2 70 MEN 3690 Pee ee pe arsed 0 250 500 750 1000 0 250 500 750 1000 FSC A FSC A Figure 2 6 Gating strategy to profile CD34 cells enriched from PBMC using the MACSQuant Analyzer A P1 region was defined to remove de
93. ature using the left mouse button or by touching the display with your fingertip Functions Statistics or Functions for each selected Feature can be displayed cv or hidden K by selecting or deselecting a Function using the left mouse button or by touching the display with your fingertip The following statistical functions may be displayed Mean StdDev standard deviation CV coefficient of variation Min minimum Max maximum Median and Modal values Table 6 10 An overview of the Properties settings for the Statistic option Overview of the Properties settings for Text A variety of display settings for the Text option can be modified using the Properties 6 i 7 option from the dropdown menu 1 Click the icon located adjacent to the Text box 2 Click Properties AS The properties window will appear Note Refer to Table 6 11 below for an overview of the various display Properties that may be modified for Text boxes using the MACSQuantify Software Tab Category Property Associated screenshot Description option Text Script Free text or a MACSQuantify Software script can Te xt Script Text be entered into the Script Text field Scripts are based on HTML hypertext mark up language and can be written to automatically display statistics about each region or gate Specific regions can be removed from or added to text table by using the Ove
94. auanaes 166 6 13 4 Post acquisition data ANIAIVSIS cissaceceneneccensnsctecenscsenensnsenensceauetensesetensnsesensanesanes 168 OT 5D TIVE GAC a os pansies wes a hae ands tam aaa vies E EI E P E A A 172 O15 6 STOPS GAGC wien EE ADIS tan sine ora penser oa andy Sua rade o here od aa pended 173 6 14 GROUPING DATA POST ACQUISITION 2ccececeececeececeeceaceceeceaeseeaeeceaeeseaseseateateseasentaseataneas 175 OLS EXPORT SAMPLE LIST oosa nreno ne Veiete weld baa aera ails Se Gai Marat easels r aer atk 176 7 Shutdown of the MACSQuant Analyzer ccccccccesceeeaeecuaeeeeeeaeeevauenuavensevansneass 179 Fe MANUALE SHUTDOWN sieetiecek coi ia ted enbeale oct utene S coerce uuaans 179 FeZ AUTOMATED SHUTDOWN wicchccarveaneieriecunie E a a 179 6 MACSOUANUEIVE SUDDONE U ssconiren enna N E EAR 181 Se Mamtena hte ser AEA 182 Oo GENERAL MAINTENANCE oostmerbsaneti na a a ones 182 I FT PURIDMIAIITCIAN CC ioiek e r ie e EEEa 182 9 12 EXCHANGING TUIG CONTATIIONS vie wires iensiiesaaseecoesieveeeednebiedaie EEE EERE 183 9 1 3 Exchanging the MACSQUANC COlUITIN visscecenenenenenecnenenenscunensnssanenesenanansnsaneneness 184 OT AEX Nang NT TUSOS initi E diane aetna heeoonn 185 9 1 5 Exchanging the hydrophobic air filtefS wessccccccccnenenenenenenenenenesesesesesessensnsnenenenenens 186 OF 0 EXCHANGING Ne PICU CCE rrna es hated A OAE niacin R 186 0 17 Cleaning tie WasSWhG SCAU ON sacs ze ee reir a T E Ara 186 9 TE GICANINIG Ne uUptake PO osere re ens anOaediens
95. bers can be entered directly into the field or by using the arrows The y and x axes scales of a dot plot and density plot or the x axis scale of a histogram can be configured as follows As required automatically configured lin linear scale log2 5 logarithmic scales hlog hyperlog scaling All regions gates are shown on the chart Only a select region gate is shown on the chart No regions gates are shown on the chart Regions gates can be displayed cw or hidden tas by selecting or deselecting a region using the left mouse button or by touching the display with your fingertip Functions for a region can be displayed W or hidden tas by selecting or deselecting the function using the left mouse button or by touching the display with your fingertip The following functions can be changed for plots and histograms Name Region name e g P1 Events in selected region are displayed as a percentage of the total number events in the current gating strategy T Events in selected region are displayed as a percentage of the total number of acquired events Count The actual number of events occurring in the selected region is displayed Table 6 8 An overview of the Properties settings for dot plots and density plots 156 Overview of the Properties settings for Histograms A variety of display settings for Histograms can be modified using
96. bes 44 3 4 Materials required for maintenance of the MACSQuant Analyzer Solutions and hardware required for maintenance of the MACSQuant Analyzer are discussed below 3 4 1 Solutions Disinfectant solution On spillage or splashing of sample it is recommended to clean the port of the automated arm and the surface of the instrument with 70 ethanol or isopropyl alcohol and a dampened tissue Alternatively use alcohol swabs Distilled water It is recommended to remove build up of salt crusts with the use of a tissue dampened with distilled water 3 4 2 Hardware MACSQuant Columns For pre enrichment of rare cells prior to analysis The MACSQuant Column has a capacity of 5x10 magnetically labeled cells The column should be replaced every three months Column substitute For installation prior to storage of the MACSQuant Analyzer when storing for longer than three months The instrument is delivered with column substitute installed Hydrophobic 0 2 um air filters Hydrophobic air filters are used to vent fluid bottles and maintain sterility Do not use hydrophilic filters since they are easily blocked upon contact with liquid Pre filter The pre filter is designed to prevent particles salt crystals etc from entering the fluidics system When installing the instrument the white lid on the top of the filter may need to be unscrewed to bleed the system 3 5 Calibration of the MACSQuant Analyzer hardware When
97. chnical support for assistance see section 1 3 Name description Position in box Fluid sensor cable module Right at the bottom of the box Pouarc Top storage compartment Fluidics tubing 6x Top storage compartment Bottle closures with sensors 4x Top storage compartment MACS MiniSampler with cover Packaged in box located in upper storage compartment Empty fluid containers with caps In bottle holders Single tube holder Top storage compartment Tube racks Packaged in box located in upper storage compartment MACSQuant Analyzer User manual software DVD keyboard and mouse Table 3 1 Inventory and location of parts within transportation box 1 Open the flight box or cardboard box not shown and remove the top layer of the packaging to reveal the instrument and associated packaging Figure 3 1 Opening the MACSQuant Analyzer flight box The lock handle must be turned 1 in order to release the lock clip 2 The MACSQuant Analyzer may also be transported in a reinforced cardboard box 32 Note The top layer holds the MACSQuant Analyzer user manual the MACS MiniSampler when included and various bags containing accessories Carefully remove these parts 2 Remove boxes containing the MACS MiniSampler and cover and the accessories Figure 3 2 Packing format of the MACSQuant Analyzer and accessories 3 Remove the foam packaging from both sides of the MACSQuant Analyzer Note Two persons are required to l
98. ck 5 4 Place the loaded rack onto the MACS MiniSampler 5 Click Start calibration The needle arm will automatically insert the needle into a tube located at rack position D6 File Edit View Mode Analysis Window Help admin E rop m p 7 i 7 7 rauan Ji a S a leal 4 T z GS gees AEA FOS EE Fe EbjsavhiOooebrisk Samples Experiment Tools Channels 5 Calibrate uptake unit H s z L Uptake unit calibration Calibrate rack detection 1 First push the Start calibration button to begin the calibration be careful the hardware moves Show backup registry 2 Move the needle and sampler by the hand to the specified position and press Save calibration r 3 Test your calibration using the Test button select position you want to test Update software Export us emove extemal media Needle arm calibration relative to w n Time and date D Zoe o Fy Stertcalibretion Save calibration Zare 0 __Statcaibrton _Sevecatbrion J Tet a Sete 0 10 la Start calibration Save calibration E sicottroion _ Figure 3 25 Calibration of the uptake unit is underway The yellow closed circle indicates that the needle arm may be freely moved for manual calibration 50 6 The needle should be positioned as follows i At the center of tube on the y axis i e equidistant from the tube edges ii Only a fraction of a millimeter from the bottom of the
99. commended to assign meaningful names in the software for each photomultipler tube detector before beginning an analysis i e the naming nomenclature must correspond to the fluorochromes used for cell staining For example FITC fluorescein isothiocyanate has an emission maximum of 521 nm in water and is thus detected in the green fluorescence channel FL2 525 nm with a bandwidth of 50 nm In contrast APC allophycocyanin with an emission maximum of 660 nm is measured in the red fluorescence channel FL6 655 730 nm The MACSQuant Analyzer is equipped with three lasers for measurement of up to seven fluorescence channels FL1 FL7 and two scatter channels FSC SSC For a list of representative fluorochromes and their respective detection channels see Table 2 1 Excitation Photomultipler tube PMT name wavelength 405 nm FL1 VioBlue 450 50 488 nm FL2 FITC 525 50 FL3 PE 585 40 488 nm FSC SSC 488 10 Table 2 1 Summary of compatible fluorochromes and respective channels 23 After antibody labeling the needle arm of the MACSQuant Analyzer draws a pre definable volume of the cell sample into the instrument Cells are either transferred directly to the flow cell for analysis or can be diverted to the MACSQuant Column for pre enrichment of magnetically labeled cells see section 6 3 6 Once in the flow cell each cell individually passes through the path of a laser and the deflection of light from the cell is used to pr
100. cured from another manufacturer Information for ordering MACSQuant Buffers can be found in Table 3 3 Description Color code Capacity Order no MACSQuant Analyzer Running Buffer Blue 6x1l 5L 130 092 747 MACSQuant Analyzer Washing Green 6x 1 5L 130 092 749 Solution MACSQuant Analyzer Storage Solution 130 092 748 MACSQuant Starting Buffer pack 130 094 190 MACSQuant Washing amp Storage 130 092 801 Solution Kit MACS Bleach Solution 130 093 663 Table 3 3 Running buffer and solutions for use with the MACSQuant Analyzer For safe operation of the MACSQuant Analyzer all fluid containers must contain at least 150 mL of the respective solutions and running buffer except for the waste container In order to prevent contamination of the fluidics system and the MACSQuant Column the use of non sterile buffers and solutions is not recommended 43 MACSQuant Analyzer Running Buffer The MACSQuant Analyzer Running Buffer is a sterile filtered ready to use buffer containing 0 09 azide as a preservative The buffer is supplied in a 1 5 L container that can be connected directly to the MACSQuant Analyzer MACSQuant Analyzer Washing Solution The MACSQuant Analyzer Washing Solution is a sterile filtered ready to use solution to rinse the fluidics system before the MACSQuant Analyzer shutdown It contains a detergent that dissociates cell aggregates and prevents the formation of plaques in the fluidics The MACSQuant
101. d for analysis 6 8 3 Rack configuration and sample grouping Sample grouping can be made before acquisition or afterwards during data analysis Refer to section 6 14 for information about sample grouping after data analysis 125 What is the benefit of sample grouping The maximum sample volume that can be acquired in a single step by the MACSQuant Analyzer is 450 uL There are occasions when the sample size is of course greater aliquots of the sample must therefore be spanned over two or more tubes By grouping these samples the acquired data will be consolidated into a single file on the hard drive which can also be analyzed in a single data file or analysis plot O 1 2 3 4 5 6 e pe e YOO Grouped BA Sill Noe Samples 600000 oe 7 can be VMY YY _ gt jm analysed O00000 cas im gt E q F gt e gt Group Three groups created analysis folders created Figure 6 25 Schematic of the grouping process To group samples 1 Using the Experiment tab select the desired rack from the drop down list Experiment Rack Chill 5 tube rack oO Single tube rack Fi Single tube rack si Chill 5 tube rack Project Chill 15 tube rack Chill 50 tube rack 96 well Description Samples 1 amp 2 were grouped Sample ID EELEE ote 2 Click to open the corresponding rack dialog box Alternatively use the keyboard shortcut Ctrl Alt R 3 Select sample po
102. d login to a user account 6 Insert the memory stick into a USB port of a personal computer with MACSQuantify Software installed 94 7 Click File and Import External media More than one external device found Please select one and press ok sw REMOVABLE D v REMOVABLE D swe REMOVABLE E Figure 5 22 If more than one external USB is attached you will be prompted to selected the correct external device 8 Use the dialog box to select the type of file for import e g Workspace Instrument settings Data files etc Note If a copy of the imported file already exists on the personal computer the following dialog box will appear Overwrite 2 File C capf user adminjdevice Cell cycle analysis cfg exists Overwrite To overwrite a single file click Yes To overwrite all files for import click All No aborts the procedure Note The imported files are copied to MACSQuantify Software It is necessary to delete files off the memory stick using windows explorer Note It is of course also possible to simply move files from the memory stick to a personal computer using windows explorer 5 10 3 To perform backup to network drive 1 Please contact your administrator if a network drive has not been configured for backup Note If a network drive is not configured the MACSQuant Analyzer software MACSQuantify will search for USB and DVD backup media instead 2 Click 3 The files will be a
103. detection class 1M laser The laser radiation from these devices is not visible Do not view directly with optical instruments e g lenses magnifying glasses or microscopes Viewing the VCSEL port within a 100 mm distance using optical instruments could be hazardous to the eye Do not intentionally direct the laser beam at others The device is also equipped with powerful LEDs for the illumination of the supply bottles and with a 2D Code Reader which uses powerful LEDs for illuminating the reading area class 1 LED Do not look directly at LED radiation or reflected LED radiation from a mirrored surface Otherwise eye injury may result Do not disassemble modify or remove the installed laser radiation sources or their mounting rackets The laser radiation sources do not automatically stop emitting when disassembled Do not allow water oil dust or other foreign substances to stick to 2D Code Reader aperture window This may cause read errors Use a soft dry cloth to wipe any substances from the scanner Do not use alcohol or other cleaning substance Radiation of disassembled units may lead to eye injuries The MACSQuant Analyzer is classified as a Class 1M laser product per standard IEC 60825 1 1993 Al 1997 A2 2001 CAUTION Use of controls or adjustments or performance of procedures other than those specified herein may result in hazardous radiation exposure ae S amp S t N Figure 1 3 Position of lasers and L
104. down list 5 1 Quick guide to the Express mode main workspace The Express mode main menu for the user Express User EU is represented below mq Express User EU ZG 0 IV UIII Help Custom Logout Definition Rack Single tube rack Filename EU2009 09 26 001 mad Sample ID il Description Mode Figure 5 1 Express mode main menu 74 Category icon Description Definition Definition is used to setup an experiment i e enter sample details such as name description and required analysis mode Acquisition Acquisition mode displays live data that is being acquired Analysis Analysis mode is used to analyze acquired data for example using an analysis template The Single tube rack Chill 5 15 or 50 tube racks and 96 well formats can be Single tube rack Chill 5 tube rack chosen using the Rack drop down list Chill 15 tube rack Chill 50 tube rack 96 well Filename N52009 01 28 001 mqd The filename is shown is this field not changeable The filename is user s initials and date followed by file number SampeiD Sample ID can be entered using this text field Description E Sample description can be entered using this text field Selecting Analysis from the Mode drop down list reveals a list of analysis templates available for immediate use These templates correspond to the MACS Mc_cOI4h Control Cocktails and count programs For more infor
105. e precision and relevance of the analyzed data is dependent on the sample size and choice of statistic respectively The MACSQuantify Software can display a summary table of statistical attributes namely count percentages arithmetic mean coefficient of variation CV minimum maximum medium median and modal statistics In addition since the MACSQuant Analyzer performs volumetric cell enumeration the actual cell count can also be displayed with each measurement Text The MACSQuantify Software Text option is a text box that may be used to enter alphanumeric characters that may be used for example to document details about the experiment the gating strategy or specific dot plot Scaling flow cytometry data using MACSQuantify Software Standard dot plots showing side scatter and forward scatter typically use linear scales This is often not possible when fluorescence labeled and non fluorescence labeled cell populations are being analyzed as the difference in fluorescence signal intensities can extend over several orders of magnitude As a consequence a logarithmic scale must be used The impact of selecting an appropriate scale is exemplified by Figure 2 4 below In this example a sample was analyzed which contained a population of white blood cells labeled with the fluorochrome phycoerythrin PE A linear scale was used to display a dot plot of forward scatter vs side scatter A and to display a histogram of PE fluorescence intensi
106. e cell analysis See www miltenyibiotec com en NN_662_MACS_Control_Antibody_ http Cocktails aspx for a list of available products No rare cella nalysis is not required 3 The Standard or Fast modes do not involve sample pre enrichment Having addressed the above questions the experiments can be easily defined using the Experim ent tab Define an experiment as follows Rack 1 2 Click the Experiment tab Select the rack type using the Rack dropdown list Experiment Samples Tools Rack Rack Single tube rack oO F Single tube rack rie reren e Chill 5 tube rack Experiment Chill 15 tube rack Sample ID Chill 50 tube rack o 96 well Description 109 3 Optional Change the filename The filename is automatically created by the MACSQuantify Software using the following nomenclature lt User initials gt lt Date VYYYY MM DD gt In this example user CU created a file on 11 May 2009 Rack Rack Single tube rack v F File CU2009 05 11 007 vja Note To change the filename deactivate the File checkbox and enter a filename into the File field Experiment 4 Enter alphanumeric text for the Sample ID and Description for example Experiment Sample ID CD34 sample one Fi Description CD34 rare cell analysis F Flow rate 5 Select a flow rate Low Medium or High Low Medium High Pickup and measure 6 Optional Click the Mix sample c
107. e guide provides and overview to the icons and layout of the MACSQuantify Software in Custom mode 6 1 1 Quick guide to the top menu bar icons File Edit view Mode Analysis Window Help olels ddid4a Js ali Onmigele lila Samples Experiment Tools Channels Experiment Rack Single tube rack m O File adm2009 09 01_2022 004 vial Project oO Sample ID Description Flow rate Llo Medium High Pickup and measure Liquid sensor C Mis sample Mode Standard Uptake volume 150 ul Eal Sample volume 200 pl Eal Annotations Autolabel Settings FL1 VioBlue FL5 PE Cy FL2 FITC FLE APC FL3 IPE FL APC Cy FL4 PIAPE Cy5 ao aiti Time Pa Acquisition mode ad Calibration ok 00 00 Figure 6 1 Screen shot of the custom mode main screen using the admin account 97 Table 6 1 Quick guide to the MACSQuantify Software top menu bar icons Description Folder icon to open Workspaces Instrument Settings Experiments Analysis templates and or Data files depending on user access rights set by the administrator Click to save Workspaces Instrument settings and Experiments depending on user access rights set by the administrator Delete a region that was created in a dot plot or histogram Draw a region in a dot plot i e
108. e instrument operator due to hazardous voltage Protective conductor terminal Symbol is attached on the inside of the instrument Warning for service personnel On supply Off supply O O PP PPP ay 1 2 Warnings and precautions The MACSQuant Analyzer is a novel computer controlled device for flow cytometry Cells isolated using renowned MACS Technology may be subsequently measured using the MACSQuant Analyzer The MACS MiniSampler connects to the MACSQuant Analyzer and thus represents a part of the instrument The MACSQuant Analyzer and the MACS MiniSampler are designed to operate safely after installation and use by trained personnel according to general safety practices and the instructions set forth in this user manual The guidelines in this section explain the potential risks associated with the operation of the instrument and provide important safety information in order to minimize these risks By carefully following the instructions you can protect yourself and the equipment from potential hazards and create a safe work environment If this instrument is used in a manner not specified by the manufacturer user safety may be compromised IMPORTANT Please read and follow all operating instructions in this user manual and pay attention to all warnings displayed on the instrument Retain this user manual and any other safety and operating instructions provided with the instrument in a place accessible to all users for futur
109. e new installation Select N when saved data files should be deleted by the new installation Select A to abort the installation 6 The program will automatically install the software using the previously selected settings On personal computer installations a software shortcut icon will be created on the desktop 4 1 1 Registering the MACSQuantify Software on a personal computer Note If your copy of MACSQuantify Software is not registered please contact Miltenyi Biotec to obtain a registration code Visit www macsquant com for more information MACS Quantify Version 2 0 aa Use Customs Password Figure 4 1 Click Register to register your name and registration code The registration code is obtained from Miltenyi Biotec 2 Enter your name and registration code as it exactly appears on the document provided by Miltenyi Biotec Use following information on http www miltenyibiotec com to register your copy of MACSQuantify Software version 1 1 0910 R Activation key 4F5E4171 Enter your name and registration key User name Registration 71 2 3 4 5 6 4 2 Updating the MACSQuantify Software on the MACSQuant Analyzer Note The software is preloaded on MACSQuant Instrument operating system embedded Microsoft Windows XP Software updates can be easily made using a USB stick Note Perform performing a software update it is highly recommended
110. e of appropriate band pass filters contamination of FITC fluorescence in the PE recording channel CH 3 is apparent The converse is also true i e bleed of PE into the FITC channel CH 2 In flow cytometry the contaminating signal or bleed is electronically subtracted by performing instrument compensation The original uncompensated density blot and corresponding compensation matrix for the acquired data is shown in Figure 3 44 PE bright cells denoted by population A show a notable fluorescence signal in the FITC channel CH 2 This should not be the case population A must move in a downward direction so that the median FITC A fluorescence intensities are identical to PE dim and PE bright cell populations 65 srv2008 12 10_2063 013 on Instrument settings Channel Compensation Matrix v FITC A 1 0e 2 1 0e1 1 0e0 1 061 1 0e2 1 03 PE A Figure 3 44 Uncompensated data density plot and corresponding compensation matrix Population A is composed of bright PE stained cells which show a significant fluorescence signal in the FITC channel CH 2 The corresponding value in the compensation matrix is 0 0 i e no Compensation was performed To compensate for bleed of PE fluorescence FL3 in the FITC channel CH 2 the compensation value for the matrix coordinate FL3 CH2 must be increased In this case the value was increased from 0 0 to 0 012 see Figure 3 45 _srv2008 12 10_2063 013
111. e reference IMPORTANT The MACSQuant Analyzer is intended for indoor use only Do not use the instrument in areas classified as hazardous locations such as oxygen laden environments Contact your local authority governing electrical power supply building constructions maintenance or safety for more information regarding the installation of the equipment If you have a serious concern regarding the safe use of your instrument please contact your authorized Miltenyi Biotec service provider or call Miltenyi Biotec Customer Service 1 3 General precautions To reduce potential risks associated with operating the MACSQuant Analyzer please observe the following general precautions Failure to observe these precautions could result in fire bodily harm and or damage to the instrument 1 3 1 Hazard of electric shock and spread of fire Hazardous voltages Risk of loss of life or severe personal injury Unplug before cleaning Do not use if device is opened or damaged if liquids have been spilled into device if objects entered device through ventilation slots Warning Electrical devices pose the risk of an electric shock To reduce the risk of an electric shock do not open any cover other than the front access covers of the MACSQuant Analyzer nor any other accessory hardware supplied by Miltenyi Biotec All other covers of the device and accessory hardware are to be removed by authorized personnel only Special care must be t
112. ed to eliminate a cell population from analysis To create a NOT gate 165 1 Draw a region or gate around the population to be excluded from analysis MiN2009 02 27_2053 005 P1 P1 P2 P3 i 14 38 T 1 0e 2 on 1 0e2 1 0e1 1 0e0 1 0e1 1 0e2 1 0e3 Figure 6 70 Region P3 was drawn using the Rectangle drawing tool 2 In the Samples menu right click on the region of interest i e in this case P3 Samples Experiment Tools Channels 17 Sample Statistic Cells live 0 0 0 5 MiN2009 02 27_2053 005 100 0 229334 Gl Pt 62 6 143622 Gc P2 60 9 139674 Bele 44 32973 3 Select Region properties from the drop down list 4 Check the box Not This region is now excluded from analysis Optional Select a color and or name the NOT gate as desired Region properties Color E custom Name Excluded from analysif Not 5 Click Apply and OK 6 13 3 Changing the properties of regions 1 Click on the region of interest 166 Note To select a region click on the Region name using the Sample menu EREE or directly click on the region geometric region displayed on the plot File Edit View Mode Analysis Window Help oga S999s4 doaS ail daisies asso stv2008 12 10_2063 015 pos SB OPI amp Op2 amp OP3 cell cycle 001 2 To change color region name and or define the region as NOT a Right click on the Region name displayed in the Sample menu b Select Region properties
113. ed with automation in mind and for use with MACS Control reagents fluorescence antibody conjugates supplied in an optimized format for the rapid flow cytometric control of cell separations performed with MACS Technology The automated uptake of samples using the needle arm permits measurement of a pre defined sample volume which in turn permits an absolute quantitation of cells ina sample According to their fluorescence labeling different cell populations can therefore also be quantified A brief summary of the main design features of the MACSQuant Analyzer is given below 2 2 1 Fluorescence cell analysis First and foremost the MACSQuant Analyzer is a flow cytometer comprising of nine optical channels for the measurement of fluorescence signals and the relative size and relative granularity of cells In conjunction with the MACS MiniSampler the automated analysis of multiple samples can be performed with ease The MACSQuantify Software performs all common functions for the presentation and statistical analysis of collected data Data can be presented as dot plots density plots histograms or Statistical tables 2 2 2 MACS Control Applications The quality of cell separations using MACS Technology can be easily and rapidly assessed by the MACSQuant Analyzer Customized measurement and analysis protocols can be created for the fluorescence analysis of cells while antibody labeling processing and sampling of cells can be p
114. emove all obvious salt crusts Use distilled or deionized water to wet the plunger and carefully push the plunger back into the syringe 4 Install the pump syringe as described in section 9 1 1 Make sure that the syringe is installed correctly into the syringe housing 5 If the leakage persists order and install either a new pump syringe Order no 130 090 339 or a new pump Seal 130 022 101 Note Depending on the level of use the pump syringe should be cleaned every 1 3 months Correct overnight and long term storage assures that no salt deposits accumulate in the pump syringe Salt deposits may cause wear of the pump seal and thus may lead to leakage The pump syringe should not run dry at any time This can damage the pump seal and thereby may lead to leakage of the pump syringe 10 1 3 Washing station overflow Please contact the Technical Service 10 1 4 MACS MiniSampler does not move properly Check whether the MiniSampler guide is inserted properly into the corresponding slot on the MACSQuant Analyzer and ensure that the cable connecting the MiniSampler to the back of the instrument is not obstructed in any way 10 1 5 Air bubbles during measurement or no events are acquired Check for leakage within the fluidics and tubing system and perform a rinse program Also ensure that the pre filter is devoid of air Ensure that the needle calibration was performed correctly Furthermore check that the tubes connected
115. enenesenenenes 117 6 7 MUETISAMPLE PROCESSING recare incu iueterheut E heir coo eee bene velo ieee eens 118 OG SCICCUING A SAIMMDI CT ACK rote aes oi E ei oe a ese eee 118 6 7 2 Configuring the sample rack for an CXPCLiINON eo casuccccccccccecececenenececeesecenenenenenenes 119 6 8 DEFINING AN EXPERIMENT WITH MULTISAMPLE PROCESSING A WORK THROUGH EXAMPLE 2000008 122 GO BACK OUNAE E E E bat ner E nee 122 6 8 2 Rack configuration and sample definition as cccccccccecenenenenensnenenenenenenenesesesesesesesens 123 6 8 3 Rack configuration and sample GOUING ruse ccccnenenenenenenenenenetetesetesesesesesesesananans 125 6 9 WORKING WITH DATA FILES IN CUSTOM MODE a iccectescsetstaei is Maine ced iedienedie ee rannen nnne 127 6 9 1 Introduction to Tle handli scrseenGie eee eae es ee 127 6 92 OCG TCS state aires ohn aaa tees ust distaste sees woes peta estat 129 0 DS SAVING THOS rna ees use iep Gar adee secu T O tie eee 131 OG DAT DOING THOS r a Seems ea enak Seb aeael oes eve cee eee 131 0 OF EXPOl TCS aae E E E E AT eee eet ete 132 6 10 DATA BACKUP AND RESTORE IN CUSTOM MODE vieieeusehsiiacs cane heteeaee eae 133 6 10 1 To perform a backup tO a rewritable DVD icscccccccnenccccecccceterensnenensesesenesesenenenes 134 6 10 2 To perform backup to a USB memory StICK ceccccacececcccncecececensnceaeneneesensnsenensnsenes 135 6 10 3 To perform backup to network drive weccccccccccucececeneneceauecceterenenenensesesesnsnsenenenes
116. ensation and Options Custom a Rack To edit the sample rack settings 99 Reagents To open the Reagents dialog box and modify reagent settings Options To modify User Experiment Instrument and Software options Only available as administrator Calibration To view and or modify instrument calibration settings View view Mode Analysis Window ae Command Description Hardware LL LL kL Hardware To view the hardware settings Experiment table comprising Fludics Sample uptake unit Lasers and detectors Camera and System settings Experiment table Provides a tabulated overview of experiment details Acquisition Annotations Autolabel and Settings Mode Analysis Window F e Dot plot Command Description Dot plot Density Click icon to change the presented data Density plot plot Histogram format into another format e g froma Statistic Text dot plot into a histogram istogram Multilayer mode View data in a multilayer format Statistic T Text Ir Multilayer mode Command Description Analysis mode Activate Analysis template tool Previous sample Scroll through analysis windows ina Next sample reverse and forward direction Window re New analysis window Command Description AE Clone window New analysis Open a new analysis window using window predefined templates Close Clone window An exact clone copy of the analysis x Close all window is made This includes ga
117. ent Tools Channels Statistic Cells live 0 0 0 sv2008 12 10_2063 015 pos 100 0 14887 PAE ooo 14887 6 13 5 Live gate What is a live gate Only events within a live gate are acquired and saved by the MACSQuantify Software SSC A 0 250 500 750 1000 FSC A Figure 6 75 In this example when using a live gate for P1 only events acquired within the region P1 will be saved Live gates are useful when large datasets are being acquired Since only dataina single gate are saved the subsequent data file is smaller As a norm however it is recommended to acquire and save all data Live gating strategies can be saved for future use as Analysis templates 172 To perform live gating 1 Click EK to open a new analysis window Choose the required plot design Create New Plot Window x 2 Define the Experiment settings as required e g uptake volume sample name flow rate labeling strategy etc Note Ensure that the Live gate option for the appropriate gate is selected in the Experiment and Settings tab Annotations Autolabel Settings Custom z O Express Instrument setting v Analysis template Cell Cycle odora cells Gate live P1 v C Events 10000 LJ Live gate was chosen for region P1 Note See section 6 3 6 for information about defining experiments refer to section 6 7 for information about defining experiments with mult
118. er switch is located at the right hand side of the instrument Figure 12 2 Several guides at the rear and sides of the instrument ease the safe connection of tubing and sensor cables Two USB ports a VGA output port an Ethernet port and two serial ports are also included The serial ports are used for attachment of the MACS MiniSampler and barcode reader MACSQuant Analyzer Fluid Containers The MACSQuant Analyzer has two orange baskets on each side of the instrument that holds the three fluid containers required for operation of the MACSQuant Analyzer which are MACSQuant Running Washing and Storage solutions A fourth in the orange buckets is reserved for the waste container The bottle closures the fluid sensor cables and the tubing connectors are all color coded and indicated by individual symbols for the specific position of each container on the MACSQuant Analyzer see section 3 2 1 for more details Single tube holder The single tube holder can be used for the analysis of individual samples Each measurement can be started using the orange button provided on the tube holder 198 MACS MiniSampler Chill Racks and MACS Reagent Rack ae j MACS Reagent Rack 4 MACS MiniSampler lid Guiding for MACS MiniSampler li Slot for MiniSampler Guiding MACS MiniSampler Cable with plug gt Cooling tube rack Chill Rack 5 Guiding for MACS MiniSampler Figure 12 5 Rear view of MACS MiniSampler with MACS Reagent R
119. erformed in an automated fashion under the control of the MACSQuantify Software Specialized MACS Control antibody cocktails 21 are also available for the multi parameter analysis of certain cell types including CD14 monocytes and CD19 B cells 2 2 3 Rare cell detection The inclusion of the MACS Enrichment Unit within the system permits the magnetic enrichment of cells 7 situ prior to fluorescence analysis The MACS Enrichment Unit and the MACSQuant Column allow the possibility to reduce the number of cells analyzed to characterize the rare cell population of interest This is particularly useful for the analysis of cells present in low abundance such as stem cells dendritic cell subsets or NK cell subsets 2 2 4 Absolute cell quantitation The MACSQuant Analyzer employs a robotic needle arm to acquire cell samples and to apply the sample into the flow cell The robotic arm provides the advantage of automation and the ability to sample a specific volume The MACSQuant Analyzer can therefore count an absolute number of cells per uL volume of sample error margin 5 This also means that multiple cell populations can be simultaneously enumerated within a sample after fluorescence staining and analysis For example the ability of the MACSQuant Analyzer to provide absolute quantitation of cell populations permits the optimized enumeration of specific cell types using pre defined software analysis protocols and specialized antibody
120. erning drawing regions with MACSQuantify Software refer to section 2 2 8 6 13 1 Drawing regions Note In the following example the IL 17 Secretion Assay Cell Enrichment and Detection Kit order no 130 094 542 was used for the analysis of viable human IL 17 secreting leukocytes from PBMCs Enriched human IL 17 secreting cells were labeled as follows IL 17 Detection Antibody PE conjugated fluorochrome CD4 marker FITC conjugated fluorochrome CD154 marker APC conjugated fluorochrome Note For background information about drawing regions gating with MACSQuantify Software refer to section 2 2 8 To create draw a region 1 Open a data file or simply draw a region around events that are being acquired by the MACSQuant Analyzer in real time e Click File Open and highlight the data file s to be analyzed Click Open Alternatively e Select the Sample tab and right click Open in the sample window Samples Experiment Tools Channels Statistic Cells Sample E Add J Import FCS file Highlight the data file s to be analyzed Click Open 2 Click KE to open an analysis window Select the desired plot layout In this case a Plot4 layout was chosen 3 Select the plot of interest A green border indicates the chosen plot 4 Double click on the opened data file this is not necessary when acquiring data in real time Samples Experiment Tools Channels Sa
121. es will be premixed by the uptake needle Custom mode is enabled A previously saved Instrument setting and Analysis template were selected for the analysis If the instrument settings option is not checked the last successful calibration will be used The events checkbox was not selected Absolute cell counting is therefore available since you will not acquire data after 10 000 events Samples in row A A1 to A4 will be measured MACS Control MC CD14 Monocyte Cocktail was selected and the vial placed on rack position one Table 6 4 Example of an experiment definition for analysis of four samples with the MACS Control CD14 Monocyte Cocktail 112 6 4 Switching to Express mode from Custom mode 1 In Custom mode click Express mode button in the top right hand of the navigation bar ccoo 0 Il ll Sooo oo090 admin E tp Neer Figure 6 12 Switching to Express mode from Custom mode 2 The MACSQuantify Software window will change to the Express mode Note If windows are active in the Custom mode e g analysis window the user will be prompted to confirm this action Click Yes to continue and No to cancel the switch Note Any active work will NOT be transferred to the Express mode All data or settings must be saved before switching to Express mode 6 5 Printing in Custom mode The MACSQuantify Software uses installed windows printer drivers to print active workspaces Note The HP U
122. ess Check radio button to setup the account for Express mode access only Custom Check radio button to setup the account for Custom mode and Express mode access Administrator Check radio button to setup the account as an administrator Access Instrument settings Using the drop down list set the user access for each of the following criteria Experiments Reagents None User access is unavailable Read User access is restricted to read files only Analysis Read amp Write Full user access is available i e read Data files and write data to this folder These options are available for Public and Private accounts Note the data files can only be stored in one location either Private or Public Password Required Activate checkbox to ensure password restricted access to the account Reset Activate checkbox to reset an associated password The user will be prompted to enter a new password at the next log in attempt Table 6 2 Creating a new user setting user properties 4 Click OK to save the new user Settings Initials Group Rights Directory Public Private Password admin adm admin AXE C cap user admin IREAD IREAd yes 2 John Smith express E C cap user john smith ierad IERAD yes Figure 6 4 An Express password protected user account was created for John Smith JS 5 To modify or delete the user account click Properties or Remove respectively 102 Note If the user is set as an Express mode user the user
123. esses 11 1 1 Fluidics components The fluid containers will be illuminated in red when empty or the waste bottle is full The waste air and sheath pump symbols are illuminated in green when they are active Active separator valves Valves 1 3 are indicated in green Defective rotary valves are indicated in red AS the system operates the valves are displaced in the live status o open c closed Hardware monitor MacsQuant SN 23 X Fluidics Sample uptake unit Lasers and detectors Camera System Fluid container Pumps Valves 1 3 Sensors Fluid container C Valves 4 6 Storage solution Separation unit l Springe drive 1 2 Waste container o fi at Figure 11 1 Real time hardware monitor of the fluid components 191 NEUE Further information Fluid container Displays buffer solution levels in real time monitor Pump monitor Displays the status of the waste W air A and fill F pumps Column monitor Displays the status of the MACSQuant Column and MACS Cell Enrichment Unit Magnet valves Displays the position of the valves for the MACSQuant monitor Analyzer Column C closed O open green in use Rotary valve Displays the position of the general fluidics system monitor valves Sensor monitor Displays the general system pressure and fluid reservoir levels Note Air pressure 200 mbar when the operating state is active Note Laser temperature Laser 488nm 37 C Laser 635 nm 25
124. eyboard Timers Acquire Text W black Export Backup Background background Colors oe Views Statistic Overlay Histogram Plot options C Multilayer mode Region functions Feature functions Figure 6 54 Changing the default Views settings for plots histograms and tables 2 Select the required view category a Statistic click checkbox to display the header for the statistic table b Overlay the user is prompted to verify deletion or overwriting of files during backup c Histogram use dropdown menus to select default values for displaying histograms Normalization Smoothing and Mode d Plot options i Data All displays all acquired events by default Percentile displays a percentile value of the total acquired events by default namely 1 2 5 10 25 50 Fixed number displays a stipulated number of acquired events by default ll Axes X Axis displays the selected axis scale by default Y Axis displays the selected axis scale by default Note It is recommended to use the default setting As required When performing data analysis users can modify the axis scale as required e Region functions select and highlight a function and using the and buttons move the region into the desired category Unused or In use 150 Options Keyboard Timers Acquire Export Backup Regions Colors Windows Templates Views Statistic Overlay Histogram Plo
125. fault Banki Bank2 Bank3 Bank4 Bank5 Set as default Mode in 3 4 Comp No Mode lin Comp No Mode log4 v Comp M Mode log4 v Comp Yes P J Mode log4 Comp Yes P Mode log Comp Vest E Mode log4 w Comp Yes v Mode Comp Yes P Mode log4 Comp Figure 3 39 Selecting channels for compensation In the above example a 4 color calibration was performed which comprised fluorochromes to test the following primary signals Fluorescence 2 FL2 FITC yellow green Fluorescence 3 FL3 PE red orange Fluorescence 4 FL4 PE Cy5 5 red Fluorescence 6 FL6 APC blue green No fluorochrome was included for evaluation of the FL5 channel PE Cy7 Compensation is nevertheless desired for this channel as indicated by Comp Yes 3 Instruct the instrument to perform automatic compensation from the Experiment tab Select the Settings tab in the lower section of the left panel and click on the Express radio button Click on the Type pull down list and select Setup Similarly choose Compensation from the Mode pull down list Samples Experiment Tools Channels Experiment Rack Single tube rack a F File adm2009 09 07 001 vfa O Project F Sample ID io Description Flow rate Low a Medium High Pickup and measure Liquid sensor C Mix sample Mode Standard
126. fault open circle indicates no operation Clear Single click on circle single Closed green circle with orange rim Sample selected for finger touch measurement Orange circle indicates that the sample is selected and any alterations made to the measurement strategy e g labeling will only apply to sample positions with this designation Double click on Closed green circle Sample selected for measurement circle double finger touch Closed blue circle Measurement in progress Closed gray circle Measurement finished Closed yellow circle Processing of sample has commenced e g sample has been labeled and incubation is underway Table 6 6 Summary of rack configurations More than one rack position can be selected at once An entire rack row or column can be selected or deselected Furthermore samples can be grouped together using the Group function After sample acquisition grouped samples can be analyzed together 120 in a single dataset analysis window Refer to Table 6 7 for information on the selection of multiple rack positions User action to select Effect Details multiple sample positions Single right click of o Use this button to change the settings for the multiple sample A yor yor all rack positions Deselect All menu button Chaer Al Clear Selected E j Note In order to set all rack positions to allow Measurement and modification of the experiment strategy e g labeling Click Select All foll
127. file paths for user groups 2 Public To change the location of the global folder click and select an appropriate file location To make a new folder click Private To change the location of the private folder click and select an appropriate file location To make a new folder click 3 Change the values between as required 4 Click Apply to implement changes Click OK close the window 139 To assign a default naming conventions to user files and folders 1 Click Edit Options and Files Options Users Files Access Experiment E Instrument Public global Software Private luser 4Date Zlnitials4 D ate Figure 6 39 Changing the default naming convention for user folders and user filenames 2 Path The default naming convention for user folders is Date i e new folders are creating according to the system date YYYY MM DD File The default naming convention for user files is lnitials Date i e new files are creating using a prefix of the users initials followed by the current system date For example filename srv2008 12 10_2063 013 ori was created by user Srv on the 10th December 2008 The remaining part of the filename was defined by the user sv2008 1 2 10_2063 01 3 ori stv2008 12 10_2063 014 neg i srv2008 12 10_2063 015 pos 3 Change the values between as required 4 Click Apply to implement changes Click OK close the window To
128. gt Start calibration Offset 13 60 44 70 0 10 mm Start calibration Save calibration Test Offset 13 90 45 30 4 00 mm Zoffset 0 Start calibration Save calibration Test Offset 11 60 45 40 4 00 mm Figure 3 22 Green closed circles indicate successful calibration of the needle arm and single tube rack Calibration of chill racks recommended if a MACS MiniSampler is attached 1 Ensure that the MACSQuant MiniSampler is correctly attached to the instrument This includes fastening of the corresponding cable to the External CAN port located at the back of the instrument see section 3 2 3 for information about correct connection of the MACS MiniSampler 49 2 Click the Experiment tab and select the Chill 5 tube rack format from the Rack pull down menu Samples Experiment Channels Experiment Rack Chill 5 tube rack O File adm2009 09 28 001 vial o Figure 3 23 Selecting Chill 5 tube rack using the Experiment tab Note The Chill 15 and Chill 50 rack do not need to be calibrated separately The calibration of the Chill 5 will also ensure for the appropriate calibration of the Chill 15 and Chill 50 rack Note If the Chill rack 5 is not selected the following error will be reported Specified rack must be specified in the experiment settings Figure 3 24 Position D6 marked X is used for calibration of Chill Ra
129. gure 6 73 Viewing results of a CD14 analysis CD14 monocytes were analyzed using an appropriate experiment design and gating strategy using the MACSQuant Analyzer and MACSQuantify Software Applying previously saved instrument settings PMT voltage and compensation settings It is possible to apply instrument settings associated with a data file To apply a previously saved instrument setting that is associated with a data file 1 Click and highlight the Instrument settings tab Highlight the desired instrument setting from a Public Private or External source Public Private wal External Directory device C CD14 Experiment 1 periments ey eagents A Analysis templates ao Setting cD1 4E xptl Le Instrument settings gt OO O Exptl 2 Click Open The file settings will be loaded 3 Open the desired data file s The files will be loaded and can be view from the Sample tab 4 Click on the Sample tab menu 170 5 Right click on the data file and select Apply instrument settings Samples Experiment Tools Channels Sample Statistic Cells live 0 0 0 RAA aera asa a srv2008 12 10_ Open Ctrl 0 srv 2008 12 10 lm F Add Import FCS file Ti Export Sample Ungroup Resample Remi Apply instrument settings Apply analysis template View with Analysis cd14 Export sample list Recompensate post acquired d
130. h on the MACSQuant Analyzer 2 Click Register on the opening login box MACS Quantify Version 2 0 User Customer User Password end Figure 4 4 Click Register to register your name and registration code The registration code is obtained from Miltenyi Biotec 3 Enter your name and registration code as it exactly appears on the document provided by Miltenyi Biotec Software registration PR Use following information on http www miltenyibiotec com to register your copy of MACS Quantity oftware version 1 1 0910 R ctivation key 4F5E 4171 Figure 4 5 Registering MACSQuantify Software 4 The instrument will startup and perform a system check 73 5 Express mode The Express mode is designed to simplify the setup running and analysis of experiments With only a few actions users with minimal flow cytometry experience can perform complex flow cytometry experiments Each user s settings are determined by the administrator at the time of the creation of the user profile Express mode users are allowed to perform only minimal alterations to settings To modify Express mode settings and or gain access to more advanced options the Custom mode must be used Note Sole Express mode users do not have permission rights to perform Calibration or Compensation These options are restricted to Administrators in Custom mode and are located under the Mode and Setup drop
131. has a barcode on the rear side that is detected upon starting the separation process 1 Remove the transparent protection foil from the lens of the rack detection Note the positions of the MACS MiniSampler guiding 2 and its corresponding slot 1 located at the front of the instrument Figure 3 9 Location of the MiniSampler guide 2 and receiving slot 1 for the MACS MiniSampler 2 Tilt the Minisampler and slide the guiding into the receiving slot until resistance is met lower the rack to a horizontal position i e the rack is locked in the position illustrated by the above figure 3 Ensure that the MiniSampler is completely inserted and secured 38 4 Note the position of the lid guiding at both sides of the MiniSampler and attach the lid e f MACS Reagent Rack 4 MACS MiniSampler lid Guiding for MACS N MiniSampler li Slot for MiniSampler MACS MiniSampler Guiding Cable with plug Cooling tube rack Chill Rack 5 Guiding for MACS MiniSampler Figure 3 10 Rear view of MACS MiniSampler with MACS Reagent Rack and Chill Rack 5 5 Place the MiniSampler cable underneath the MACSQuant Analyzer and connect it to the socket 4 labeled External CAN at the rear panel of the instrument Socket for sensor cables Socket for code reader Socket for MACS MiniSampler Socket for main plug Figure 3 11 The MACS MiniSampler cable is attached to socket 4 at the back of the instrument 3 2 4 Pos
132. he fuse as instructed by the Miltenyi Biotec technical support return the fuse holder to the housing and plug in the main power cord 9 1 5 Exchanging the hydrophobic air filters Hydrophobic air filters are attached to the bottle closures to vent the liquid bottles To avoid clogging of the filters and to prevent contamination of the liquids the air filters should be exchanged if they come into contact with liquid They also should be exchanged once a year to avoid clogging through the deposition of dust 9 1 6 Exchanging the pre filter The sheath particle filter provides a physical barrier to prevent debris larger than 70 um in size from entering the fluidics system from the fluid containers The filter can be easily exchanged when blocked or at regular intervals to prevent blockages 1 Shut down the fluidics system and remove the power cable from its socket 2 Place a dish underneath the sheath filter to collect liquid 3 Remove the filter unit from its holder and disconnect the tubing 4 Connect a new filter to the tubing and reattach the filter to its holder 9 1 7 Cleaning the washing station 1 Remove single tube holder if needed 2 Open the front cover to the right side 3 Open the cover of the washing station to the left side Note Potentially contaminated liquid may spill out of the orifice of the washing station and the tubing Therefore wear protective gloves protective clothing and safety glasses to avoid contact
133. he instrument should only be operated from a power source indicated on the product s electrical ratings label If you have questions about the type of power source to use contact your authorized Miltenyi Biotec service provider or local power company Do not use extension cords or power strips Do not overload an electrical outlet The overall system load must not exceed 80 of the branch circuit rating 1 4 6 Accessibility Make sure that the main switch as well as the connector for the power cable are easily accessible and located as close to the operator of the instrument as possible If it is necessary to disconnect the power supply unplug the cable from the power outlet 1 4 7 Peripheral devices Only original MACSQuant Analyzer Equipment shall be attached to the connectors labeled External CAN CAN1 and CAN2 The voltage levels on these connectors shall not exceed hazardous voltage levels of 30 Vrms and 42 4 Vpeak or 60 Vdc Only the MACSQuant Analyzer Bottle Sensor Cable should be attached to the Bottle Sensor connector Only a 2D code reader recommended by Miltenyi Biotec should be connected to the RS232 BCR connector External laser devices connected to the connector labeled RS232 BCR have to comply with the standard IEC 60825 1 External computing devices connected to the R232 interface connectors labeled COM 1 have to be listed in accordance to the standard UL 60950 1 Only use connector cables
134. he lower dropdown box select the desired template aoe Description CT Mode Note Contact your MACSQuant Analyzer administrator if the desired template is not available Templates can only be created and managed by administrators or Custom Selecting an analysis mode 1 Select Analysis from the Mode dropdown menu e EME 2 From the lower dropdown box select the desired Analysis criterion Corresponding graphic Option Description count To perform absolute cell counting Mi _CD14_h McC CD19_h MC_CD14_h Evaluation of MACS Cell Separations using CD14 Mc CO34 h MicroBeads human or the Monocyte Isolation Kit Il MC _CD3_h human order number 130 092 859 ee MC_CD_19_h Evaluation of MACS Cell Separations using the B Cell Isolation Kit human order number 130 092 860 MC_CD_34_h Evaluation of MACS Control MC CD34 Stem Cell Cocktail human order number 130 093 427 MC_CD3_h Evaluation of MACS Cell Separations using the Pan T Cell Isolation Kit Il human or CD3 MicroBeads human order number 130 092 881 MC_CD4_h Evaluation of MACS Cell Separations using the CD4 T Cell Isolation Kit Il human or CD4 MicroBeads human order number 130 092 914 MC_CD8_h Evaluation of MACS Cell Separations using the CD8 T Cell Isolation Kit human or CD8 MicroBeads human order number 130 092 912 Table 5 5 Example of options available for performing cell analysis in Express mode 3 Click Start Measuremen
135. he single tube holder and dispense one drop of MACSQuant Calibration Beads into it Note Ensure that you mix the calibration beads prior to dispensing 4 Click OK to commence the calibration process The uptake needle will dilute the calibration beads to a total volume of 0 5 mL following which 100 uL will be taken up and injected into the flow sheath port for the calibration procedure During calibration the gain and trigger for each respective channel will be automatically adjusted The MACSQuant Calibration Beads consist of two sizes of beads 2 um unstained beads and 3 um beads stained with fluorochromes to emit fluorescence in all 7 channels For more information about the MACSQuant Calibration Beads please review the product data sheet available at www miltenyibiotec com File Edit View Mode Analysis Window Help admin Bae 43 Gel eal it mm ca el es 5 lO Singe tube rack _ ac adm2009 09 01_2022 003 SA Project Sample ID Description Flow rate Medium Pickup and measure Liquid sensor C Mix sample Mode Standard Uptake volume Sample volume Figure 3 35 Calibration is underway When the process is successfully completed the MACSQuant Analyzer Status bar should report MACSQuant ready Calibration OK These settings will be automatically saved as the default settings 5 The calibration results for each channel
136. heckbox L Mix sample to premix the sample before sample pickup data acquisition and analysis 7 Select an analysis mode from the Mode dropdown list Standard v Enrich Measure Ori Neg Pos EnrichS Measure Ori Neg Pos Enrich Measure Pos EnrichS Measure Pos See Table 6 3 for details about each option Uptake volume a i 50 ul 8 Enter the Uptake volume and Sample volume vie soou A maximum sample volume of 5 mL and a maximum uptake volume of 450 uL can be entered Annotations tab 9 Optional If required modify the annotations for the fluorescence channels The default settings are shown below Annotations Autolabel Settings FL1 CD45 VioBlue FL5 PE Cy FL2 FITC FL6 CD15 4PC FL3 CD14 PE FL APC Cy7 FL4 PI Autolabel tab 10 Optional If autolabeling is required add the relevant reagents by clicking on an lt add gt checkbox Annotations Autolabel Settings C lt add gt C lt add gt C lt add gt C lt add gt C lt add gt C lt add gt C lt add gt C lt add gt C lt add gt C lt add gt 110 Peeererr errr rrr Click an available checkbox Select the reagent reagent rack position incubation time titer and order from the dropdown lists Reagents Category Reagent RI Human PE CD45 PE human B Calibration MACSQuant Calibration Beads abg 5mL MACSQuant Calibrati
137. hlight the file location Private Public or External Ope 3 Select the file type and click Open oen To open reagents 1 Highlight the Reagents tab on the Open window 2 Highlight the file location Private Public or External To open analysis templates 1 Highlight the Data files tab on the Open window 2 Highlight the file location Private Public or External 3 Select the file type and click Open Loren Pic Private Esera ciete Oreo To open data files 1 Highlight the Data files tab on the Open window 2 Highlight the file location Private Public or External 130 3 Select the file type and click Open Loren am Petcu Private cote Droctoy Da 6 9 3 Saving files 1 Click to open the Save window Figure 6 31 The Save window Custom users and administrators are able to save Workspaces Instrument settings Experiments Reagents and Analysis templates Analysis templates can only be saved when the Analysis mode icon Al is activated 2 Click on the Workspace tab Instrument settings tab Experiments tab Reagents tab or Analysis template tab to save the relevant file type Figure 6 32 Saving file types Left Workspaces Instrument settings Experiments or Reagents file types can be saved Right Analysis mode was activated All in order to save Analysis templates 3 Using the keyboard enter the file name 4 Click Save to save files
138. ially available flow cytometers makes the instrument ideally suited to benchtop operation within the laboratory Also the instrument has several design features that permit the fully automated processing of cell samples from sample labeling and mixing through uptake and magnetic enrichment to fluorescence analysis The MACSQuant Analyzer can be optionally fitted with the MACS MiniSampler The MiniSampler is a motorized sample rack holder that can hold tube racks of varying formats including 96 well microtiter plates The fully automated uptake and processing of multiple samples is possible under control of the MACSQuantify Software thus permitting the user a hands free high throughput operation Automated maintenance procedures are also a design feature of the MACSQuant Analyzer This includes different system wash programs before each measurement automatic priming of the instrument and programs for shutting down the instrument for overnight or long term storage After sample uptake the instrument can analyze fluorescence labeled cells using up to nine optical parameters seven fluorescence and two scatter channels The software provided with the MACSQuant Analyzer can perform standard flow cytometric data analyses and illustrations including histograms dot plots density plots gating and Statistical views Data can also be collected in terms of time area height and width During acquisition the data are automatically sto
139. ible with most data handling software Options Linear All data will be saved in linear format i e without logarithmic manipulation Add ext info Information about the file format time data and file type is added to a text header of the data file As this information varies according to the size of the data file the text header may also vary in size Some non Miltenyi Biotec flow cytometry data handling software are unable to work with files which have a larger info text header It is therefore recommended to disable this function by default Compatible Export data for use with other flow cytometry analysis software for example FlowJo In actual fact data is exported as Float and Linear data 3 Click Apply to implement changes Click OK close the window 146 To assign default settings for Backup 1 Click Edit Options Software and Backup E options Users Experiment Instrument Software Files Keyboard Timers Acquire Backup Backup Always data files v Deletion Automatic Ask H nese Sn a ae Windows 4 Views Figure 6 50 Changing the default file backup settings 2 Use the dropdown list and or radio buttons to activate deactivate a feature Files Backup a always ask before performing a backup the user is prompted to verify which file types are to be backed up b always all files all files are always backed up there is no user prompt to ve
140. ift the MACSQuant Analyzer The instrument must be gripped at the base of the orange bottle baskets located at both sides of the device Note that the instrument is heavier at the front Ensure the front of the instrument is stabilized while lifting it Due care must be taken while lifting the MACSQuant Analyzer Miltenyi Biotec accepts no liability for potential injuries sustained during lifting and or movement of the device 4 Place the instrument onto a stable worktop surface e g laboratory bench Remove the plastic bag surrounding the device Note Take into consideration that the instrument requires adequate air circulation for heat exchange and cooling Refer to section 1 4 2 for elaboration 33 5 Carefully remove the uptake port needle from the foam packaging Figure 3 3 Left The MACSQuant Analyzer was securely placed on stable worktop Right The plastic bag was removed Note that the uptake needle is supported by foam 6 Place the uptake port needle into its guiding at the needle arm Tubing can move Note Ensure that the tubing connected to the uptake port needle can move freely when the needle arm extends or when the needle moves into the sample uptake position 7 Adjust the angle of the touchscreen in an upright position in order to access the solution bottles and the fluidic ports on the back of the instrument 3 2 Installation of the MACSQuant Analyzer Before installation carefully read the cha
141. ight Workspaces top left Instrument settings top right or Experiments bottom left to save the desired file type To save a workspace 1 Highlight the Workspace tab on the Save window 2 Highlight the desired file location Private Public or External 3 By default the workspace will be saved to the user s private folder To save to an external drive highlight the External tab E snawi 85 Note If no external media is attached to the MACSQuant Analyzer or personal computer USB port the follow error will be reported External i i No external media Found 4 Enter the filename in the Setting field and click Save e J To save an experiment definition 1 Highlight the Experiment tab on the Save window 2 Highlight the desired file location Private Public or External 3 By default the experiment definition will be saved to the user s private folder To save to an external drive highlight the External tab Femail Note If no external media is attached to the MACSQuant Analyzer or personal computer USB port the following error will be reported External i No external media Found 4 Enter the filename in the Experiment field and click Save 21 5 7 Defining an experiment in Express mode A work through example In the following example three samples were placed in rack positions Al A2 and A3 of a Chill 5 tube rack It is intended that sample positions Al and A2 will be ana
142. ign for strong permanent magnet 1 3 3 Hazard of crushing and shearing Moving parts stay clear Hazard of puncturing or crushing fingers Operate only with attached needle arm guard CAUTION Do not open the front access covers while the device is in operation Do not obstruct the movement of the automated arm and accessory hardware during operation Keep fingers etc away from all moving parts of the MACSQuant Analyzer and accessory hardware to avoid crushing or shearing injuries or damage to the device Do not touch fluid pumps or adjust the tubing while the device is in operation Always switch off the device before adjusting any part of the fluidic system Always stop or abort a procedure before handling accessory hardware e g MACS MiniSampler or loading removing tubes from the tube rack placed on the sampler Do not circumvent any safety measures or devices Figure 1 2 Open circle shows warning sign for hazard of crushing and shearing 1 3 4 Laser and LED radiation The MACSQuant Analyzer is equipped with continuous wave lasers vertical cavity surface emitting lasers VCSEL and powerful light emitting diodes LED Warning The device is equipped with up to three continuous wave lasers class 3B laser for fluorochrome excitation These lasers are secured by protective housing Do not remove the protective housing Otherwise eye injury may result The device is also equipped with four VCSELs for automated rack
143. in Data analysis mode the instrument can only analyze data The instrument must be placed in acquisition mode to perform a measurement Data analysis mode ar Time Volume Rate Count ai oo ow Osc 0o Figure 6 7 Instrument is in Data analysis mode Green MACSQuant in Acquisition mode calibration was successfully performed one day ago time in days since the last calibration is indicated Acquisition mode ar Time Volume Rate Count 2 0 0 3 Calibration ok 1 day old ooo ow sd mse paag Figure 6 8 Instrument is in Acquisition mode Yellow Cleaning and priming of MACSQuant Analyzer instrument is not available for measurement cleaning in progress Cleaning MACS Quant ear Time Volume Rate Count Figure 6 9 Instrument is in the process of being cleaned Grey Instrument is being initialized instrument is not available 15 09 Initializing MACSQuant Figure 6 10 Instrument is unavailable Blue MACSQuant Analyzer is processing a sample Instrument is currently processing a sample Processing rack ar Measuring sample Volume Rate Count C 18 15 pie Poll 00 23 90 wl 1 414 p Figure 6 11 Sample processing in progress Note Upon completion of the initialization process the MACSQuant Analyzer is in the Data analysis mode until the instrument is placed in Acquisition mode 106 Checking the instrument status using the bottle LEDs The MACSQuan
144. incubate for 10 minutes in the dark at 4 C Wash cells by adding 1 2 mL of buffer to each tube and centrifuge at 300xg for 10 minutes Aspirate supernatant Resuspend cell pellets to a concentration of 1x10 mL Proceed to performing automated 7 color compensation on the MACSQuant Analyzer 7 color compensation on the MACSQuant Analyzer 8 To open a Saved instrument setting click File Open and highlight the tab Instrument settings Select the appropriate file 67 Note It is not necessary to open a pre saved instrument setting It is also possible to use the current settings In this case ignore step 8 9 Select a Chill Rack for example Chill Rack 5 This should be pre cooled to 4 oC 10 Position each of the seven tubes with single stained cells in columns along the Chill Rack 5 the order should be as follows VioBlue A1 FITC B1 PE C1 PE Cy5 D1 PE Cy7 A2 APC B2 APC Cy7 C2 Append the remaining two tubes containing PI stained cells D2 and unstained cells A3 at the end 11 Group the samples together to form a single group as shown below Mew Mode Analyses Window Help admin HOOD alae ANNE OE E Se Figure 3 46 Cell samples were grouped together 12 Click PJ to begin compensation 13 The user will be prompted to draw a region of interest around a particular cell population Fig Edt Yew Mode Analysis Window Help amaes Erperiman Tods Channel i
145. indow Help FEOOERErreRGSANE admin Oj Sle lt a Samples Experiment Tools Channels Ee Rack Single tube rack a W Fi File adm2009 09 01_2022 001 a Project Fi Sample ID l Description Flow rate wo Main Instrument Control X Medium High ea Switch into the acquistion mode ickup and measure Liquid sensor Mode Uptake volume Sample volume C Mix sample Standard Custom Express C Instrument setting Setup 20090828 Default C Analysis template m 100 pl E 200 wl Annotations Autolabel Settings Acquisition mode Data analysis mode C Gate C Events 10000 Ga 3 Data analysis mode Time ells ee Count 13 02 00 00 Dyl O sec l 0 Figure 6 6 Switching the instrument into acquisition mode After the instrument has been primed and all system checks have been performed successfully the instrument status and LEDs will display green see section 6 3 5 If the system status is red act in accordance with the error message The system status will prompt you if the instrument needs to be calibrated and how many days since the last calibration If the LEDs are red or if you suspect one of the solutions is too low you can replace the appropriate solution To replace a solution place the closed bottle into
146. ion Sove calibration _ EErEE ETTE San caioreson J _Sevecaibreton J C _Startcalibration Save calibration Test Note The red closed circles shown on Figure 3 16 indicate that all four uptake components Needle arm Single tube rack Chill 5 tube rack 96 well plate Reagent Rack 4 are not calibrated Note In order to meet the minimal hardware calibration requirements of the MACSQuant Analyzer the position of the needle arm in relation to the washing Station and single tube rack must be calibrated THIS IS A MINIMUM REQUIREMENT Calibration of needle arm The needle arm moves between the samples and sample injection port needle wash Station along the y and z axis It is imperative that the arm is correctly calibrated 1 Ensure that the needle arm can freely move and that no object is obstructing it 2 Click Start calibration under the heading Needle arm calibration relative to washing station The needle arm will move toward the wash station y axis before being inserted into the sheath flow port z axis Note If the calibration unit is grossly misaligned the needle may pop out of the needle arm holder This will cause a system failure but will not damage the resulting components Simply reinsert the needle into the holder and adjust the needle arm appropriately A dialog box will appear to reinitialize press ok f rile fal Epress elsa a a rmm O
147. isample processing 3 Ensure that the correct instrument settings are loaded and that compensation is correctly performed Note See section 3 6 for information about performing instrument calibration see section 3 7 for information about performing compensation 4 Ensure that enough sample reagents and buffers are provided 5 Click on the Start Measurement button gt The MACSQuant Analyzer will commence sample uptake and measurement 6 Draw regions on the plots as described in section 6 13 7 Save the Analysis template for future use 6 13 6 Stop gate What is a stop gate Unlike with a live gate all data are acquired and saved by the so called Stop gate However a Stop gate used in combination with the Events option instructs the MACSQuantify Software to acquire data from the entire analysis window until a pre 173 defined number of events are acquired within the Stop gate i e a gate or region that is defined as the Stop gate Annotations Custom Gate Event SIYLVUUDI US It CUuso UucT Autolabel Settings 750 C Express Instrument setting CD14 Experiment Analysis template Cell Cycle odora cels z stop P1 bal 250 10000 L I 0 0 250 500 750 1000 FSC A Figure 6 76 Region P1 was defined as a Stop Gate When 1 000 events are acquired within region P1 data acquisition will automatically stop and all acquired data i e the entire analysis window will be saved Stop
148. ition 2 Click Edit and Reagents Note It is also possible to activate the reagent management window by checking an Autolabel lt add gt box located in the Experiment window _ _ _ ___ _ Arnor Aatoladel Settings 3 Check the desired reagent position R1 R2 R3 R4 S1 or S2 This must correspond to the correct position on the reagent rack 4 Highlight a category that corresponds to the desired reagent using the Category dropdown list 116 5 Highlight the desired reagent from the Reagent dropdown list 6 Modify the incubation time Time label sample titer Titer and order if required 7 Click Apply to apply changes and close the window 6 6 3 Scanning reagents with the 2D code reader To enter regents manually into the reagents database The 2D code reader barcode reader is used to scan reagent vials Reagent vials are automatically recognized and logged by the MACSQuantify Software To scan reagents perform the following rm 1 Click the activate code reader icon The code reader will being blinking 2 Present the reagent vial in front of the 2D code reader Ensure the 2D code is facing the blinking code reader light The optimal reading distance is 0 5 2 5 cm from the code reader cover tilt the vial as depicted in Figure 6 16 a Figure 6 16 Scanning a reagent using the MACSQuant Analyzer 2D code reader 3 Scanned reagents are reported a MACSQuantify Soft
149. itioning of cooling tube racks and the MACS Reagent Rack 4 1 Open the lid of the MACS MiniSampler 2 Secure the MACS Reagent Rack 4 onto the MiniSampler into the left recess The engagement hook has to snap into the undercut Figure 3 12 To remove the MACS Reagent Rack gently press the rack in the planar direction 1 followed by lifting the rack in an upwards direction 2 39 3 Set a cool tube rack e g Chill Rack 5 onto the MiniSampler into the right recess ensuring that the rack barcode is facing the MACSQuant Analyzer Figure 3 13 Positioning the Chill Rack 5 adjacent to a MACS Reagent Rack 4 on the MACS MiniSampler Note Racks can be pre cooled for 3 4 hours at 2 8 C Do not cool below 0 C since Samples may freeze If recognition of the tube rack fails the instrument will display a screen for manual selection of the tube rack Before confirming the choice ensure that the rack is placed correctly into the recess 3 2 5 Switching ON OFF the MACSQuant Analyzer The main power switch is located on the right side of the instrument in front of the container baskets I indicates On O indicates Off Switch on the MACSQuant Analyzer On off switch Figure 3 14 Location of the on off switch 3 2 6 Installation of the MACSQuant Column optional The MACSQuant Column can be ordered separately and can provide the flow user a fast and reproducible way to analyze rare cells with
150. kits For example using the CD4 RTE Enumeration Kit 130 092 055 the enumeration of CD4 recent thymic emigrant RTE cells is possible from whole blood samples or peripheral blood mononuclear cells PBMCs Furthermore the automated processing of cells and the use of the MACS Mini Sampler facilitate the seamless incorporation of the MACSQuant Analyzer into routine high throughput laboratory cell enumeration procedures Labeling reagents and software analysis can be customized to suit individual applications 2 2 5 Automated cell labeling and analysis Automation of cell sampling and analysis can be extended to include the labeling of cells with the MACSQuant Analyzer With the use of the optional MACS MiniSampler the automated processing of up to 96 samples is possible facilitating the integration of the instrument into high throughput procedures 2 2 6 Flow cytometry an introduction Any given cell population can be defined by its individual expression profile of both intracellular and extracellular antigens These antigens can therefore be targeted for their detection and further analysis using flow cytometry Flow cytometers detect cells according to two basic parameters light scatter and fluorescence Cell size and granularity are inherent characteristics of a cell and vary from one cell type to another these properties are measured using the forward scatter FSC and side scatter SSC channels respectively 22 However wi
151. l human MC CD3 Pan T Cell Cocktail human MC CD34 CD133 Cocktail human MC CD34 CD133 Control Cocktail human MC CDS T Cell Cocktail human MC CD4 T Cell Cocktail human MC CD34 Stem Cell Cocktail human e Time For autolabeling an incubation time is given The recommended incubation time is automatically shown in a black font type Experienced users may wish to change the incubation time using the adjacent arrows l note that non recommended times will appear in a red font type e g EINGI e Titer For autolabeling a recommended label to sample titer is given The recommended titer is automatically shown in a black font type Experienced users may wish to change the titers using the adjacent arrows l note that non recommended titers will appear in a red font type e g Kn e Order Signifies the order at which this reagent will be used during cell processing a Further information about the reagent is shown when this icon is activated 6 6 2 Selecting and assigning reagents manually using the MACSQuantify Reagents dialog box Note It is recommended to use the barcode reader to scan reagents see section 6 6 3 This protects the user against making incorrect reagent entries However if the reagent label or datasheet insert is damaged it may be necessary to manually select reagents using the Reagents dialog box To select regents manually 1 Place a reagent onto the reagent rack noting its pos
152. le tube holder from the front of the instrument 3 Remove the single tube holder by pinching the single tube rack using both index fingers the orange button is used only for acquisition it does not release the single tube rack from the instrument The single tube rack can be easily removed by gently pulling the component away from its docking adaptor Figure 3 30 Removing he single tube holder The orange button is for acquisition only 4 The software will then direct you to re connect the single tube rack This is performed by inserting the two male pins of the single sample rack into the female docking adaptor until its clicks into position 5 Lastly the software will prompt you to press and hold the orange sample acquisition button until the calibration is completed A successful calibration is 54 indicated by a green closed circle NE Ale e S ee tout D Rack detection calibration equirements Single tube rack and Chill rack ollow on the instruction in the specified calibration on the right side Single tube rack and acquisition button detection Start calibration Calibration done Rack detection push Start calibration button to begin the calibration helen cetection Start calibration Alen Star oal ar ation uation ta bein tie caii Note For the rack calibrations the user must perform the prompted tasks promptly in order to calibrate the racks properly O
153. lor touchscreen or via an externally connected keyboard and mouse The computer is integrated within the MACSQuant Analyzer and any software updates can be easily performed using the DVD RW drive which is located at the top of the touchscreen monitor Figure 12 1 The computer operating system is Microsoft Windows XP embedded and the all cell analysis and pre enrichment programs are controlled by the MACSQuantify Software Automated arm with sample uptake port The robotic arm Figure 12 1 is a computer controlled component of the MACSQuant Analyzer fluidics system It consists of two motors that drive the arm in y and z directions and a needle for sample uptake The needle and uptake port are automatically washed in the MACSQuant Analyzer washing station during and after cell processing to prevent cross contamination between samples 195 Touchscreen Robotic arm Front cover Bottle closures 4 containers 4 Fluid containers 4 Code reader Rack detector Figure 12 1 Front view of MACSQuant Analyzer Robotic arm Needle arm Uptake port Onoff switch MACS MiniSampler LEFT RIGHT Figure 12 2 Left and Right views of the MACSQuant Analyzer Access covers The hinged front cover Figure 12 1 can be opened to the right to allow access to the components of the fluidic system including the syringe pumps the MACSQuant Analyzer Enrichment Unit and MACSQuant Column or substitute column and the up
154. ls Conversions C Convert decimal point to comma C Transpose rows and columns C Reverse samples Options Clipboard Check box to export data to the windows clipboard File Check box to export data to a Microsoft Excel file xls Location Files can be saved to a Public or Private location Name f File is checked enter the filename here taking care not to delete the Excel file extension xls Conversations the respective boxes to Convert comma to point in some languages numbers the decimal point is actually shown as a comma 177 b Transpose rows and columns the columns and rows of the export table e g Excel sheet are inverted c Reverse samples the order of the samples e g 1 2 3 etc are reversed Region functions Export sample list rv2009 02 04_2062 054 pos rv2009 02 04_2062 054 po 1v2009 02 04_2062 054 po rv2009 02 04_2062 054 po 1 2009 02 04_2062 053 neg Count ml rv2009 02 04_2062 053 ne st 2009 02 04_2062 053 ne 1v2009 02 04_2062 053 ne rv2009 02 04_2062 052 ori s1v2009 02 04_2062 052 ori stv2009 02 04_2062 052 of stv2009 02 04_2062 052 of SSSSSSSSSSSS SE Select the gates regions for export using the Regions box lv For export X Not for export Similarly use the Functions box to select deselect region functions for export Feature functions Export sample
155. ls being analyzed Yes rare cell analysis is required 1 Rare cells can be automatically magnetically labeled and enriched by the MACS Cell Enrichment Unit Depending on the selected analysis mode the enriched and non enriched fractions can be subsequently analyzed by flow cytometry A MACS Column must be installed for pre enrichment 108 Cell separation olume of was buffer used Enrich Measure Ori Neg Pos 1 Original cell fraction ori 2 Enriched target cells enrich fraction 3 Remaining non target cells or negative fraction neg 0 5 mL min h 3 00 mL EnrichS Measure Ori Neg Pos Enrich Measure Pos rate The following cell fractions are analyzed by flow cytometry 1 Original cell fraction ori 2 Enriched target cells enrich fraction enrich fraction 3 Remaining non target cells or negative fraction neg 1 Enriched target cells EnrichS Measure Pos rate The following cell fractions are analyzed by flow cytometry 1 Enriched target cells enrich fraction 0 5 mL min 0 5 mL min 0 5 mL min 4 66 mL 3 00 mL 4 66 mL Rate of elution 150 mL min 37 5 mL min 50 mL min 37 5 mL min E 450 uL 450 uL 450 uL Table 6 3 Specifications for sample pre enrichment using the MACS Cell Enrichment Unit in combination with the MACSQuant Column 2 MACS Control Antibody Cocktails are available for automated and reliable rar
156. lyzed using the MACS Control CD14 Monocyte Cocktail Sample position A3 will be analyzed using the MACS Control CD4 T Cell Cocktail Note Ensure that the instrument is primed and calibrated Check that adequate reagents and buffer volumes are provided Ensure that the Definition tab is activated Definition 86 1 Select Chill 5 tube rack from the Rack dropdown menu 2 3 4 mq Express User a le x 5 A im amp O EU Help Custom Logout Definition Fenere smeo OOE Mode Figure 5 12 Selecting the Chill 5 tube rack Left click once on rack coordinates Al and A2 Figure 5 13 Measure and select The settings for sample positions A1 and A2 may be modified e g a labeling strategy may be applied Select the Analysis Mode and MC_CD14_h from the lower dropdown list Mode Figure 5 14 CD14 MACS Control Cocktail analysis is applied to rack positions Al and A2 Use the Sample ID and Description fields to enter relevant sample information Description PMBC Preparation Figure 5 15 The above information is now associated with sample positions Al and A2 87 5 6 7 8 9 Select position A3 Figure 5 16 Position A3 is selected In doing so positions Al and A2 are automatically deselected Select the Analysis Mode and MC_CD4_h from the lower dropdown list Use the Sample ID and Description fields to enter relevant sample inf
157. mation on the MACS Control Cocktails please see www miltenyibiotec com Selecting Setup from the Mode drop down list reveals three options for instrument setup Calibration Compensation and Compensation7Colors Calibration v a NOTE Setup is only available to administrators and Custom users Compensation Compensation Colors Initials of user in the top left corner in this example Express User EU Folder icon to open Workspaces Instrument Settings Experiments Analysis templates and or Data files depending on user access rights set by the administrator Click to save Workspaces Instrument settings Experiments and Analysis templates depending on user access rights set by the administrator Print Backup data to DVD or initiate data transfer to USB or network location Activate the 2D code barcode scanner Activate touch screen keyboard Open help file Custom Switch to Custom mode Only available to users with Custom or Administrator rights L Click to logout from the session ogout l Main instrument control Click to switch between Acquisition mode Data analysis mode or Instrument off Table 5 1 Express interface icons with brief explanation 75 5 2 Login to Express mode 1 Select your user name from the dropdown list and enter the appropriate password if required 2 Click login to proceed Note If the user has been registered as an Express mode user the MACSQuantify Software
158. may also be displayed ina textbox Refer to section 2 2 7 for an explanation of these formats Note For information about file handling in Custom mode e g opening and saving data files refer to section 6 9 1 Custom mode users can change the properties of a dot plot density plot histogram and statistic or text box as follows 1 Click on the icon _ located beside the dot plot histogram or text table A popup menu will appear ls Dot plot Density plot Ne Histogram Statistic T Text Ir Multilayer mode m Properties Figure 6 60 i Popup menu 154 2 Click Properties m e l The properties window will appear Figure 6 61 Properties window 3 Select the desired property and modify accordingly For example to change the axes of a dot plot use the Axes dropdown menu E Properties PR z 1000 via lt gt Note The Properties window for Dot plots and Density plots is identical The Properties window for Histogram Text and Statistic are different Refer to the following sections for an overview of the settings available for each chart type Plots Overview of the Properties settings for Dot plots and Density plots Histograms Overview of the Properties settings for Histograms Statistics Overview of the Properties settings for the Statistic option Text Overview of the Properties settings for Text 1
159. mple Statistic Cells live 0 0 0 MiN2009 02 27_2053 021 guilty 73463 __ gt Figure 6 64 Left The data file izi was opened in the pre selected plot window middle Right Double clicking on the middle plot window enlarges the plot to cover the entire analysis window 5 Use the icons to select a geometrical shape for gating o mie fF F Note or Interval can be only used for Histogram analysis Interval is a marker or region that can be drawn on histograms in order to calculate statistics for that particular region 6 Axis forward scatter versus side scatter A region was drawn P1 using the polygon tool to exclude unwanted 161 7 debris and select for lymphocytes MiN2009 02 27_2053 021 1000 a 750 500 SSC A 2504 72 le 2 le 1 lel le2 1e3 le CD4 FITC A Figure 6 65 A polygon region P1 was drawn to exclude unwanted debris and select for CD4 lymphocytes Axis Anti IL 17 PE versus PI PE Cy5 A polygon region was drawn P2 using the polygon tool to exclude unwanted dead cells id MiN2009 02 27_2053 021 P1 e R 1e2 lel ie PIZPE Cy5 A le 2 1e 1 1e0 lel 1e2 1e3 Anti IL 17 PE A Figure 6 66 A polygon region P1 P2 was drawn to exclude unwanted dead cells 8 The region P2 was only displayed on the third plot Note Click on the plot header to select a region to display MiN2009 02 27_2053 021 P1 Click to switch o
160. n be saved to a Public Private or External file location by all users MACSQuant Data MQD is the standard file handling format however the MACSQuantify Software can also import Flow Cytometry Standard FCS file types 5 6 2 Opening files 1 Click D to open the Open window Figure 5 8 Only Experiments or Data files may by opened by Express mode users Custom mode users and administrators are able to open Workspaces Instrument settings and Analysis templates in custom mode 2 Click on the Experiment tab or Data file tab to open an experiment definition or data files respectively Figure 5 9 Highlight the Experiment left or Data files right tabs to open the desired file type 84 To open experiment definitions 1 Highlight the Experiment tab on the Open window 2 Highlight the file location Private Public or External 3 Select the file type and click Open Les To open data files 1 Highlight the Data files tab on the Open window 2 Highlight the file location Private Public or External 3 Select the file type and click Open L 5 6 3 Saving files 1 e to open the Save window Figure 5 10 The Save window All users are able to save experiment descriptions instrument settings and workspaces 2 Click on the Experiment tab Instrument settings tab or Workspace tab to save the relevant file type Figure 5 11 Highl
161. n button detection Start calibration Calibration done Rack detection Calieration done Ghelen detection Start calibration Jan Sia saliai Stele te Jaeja saligi 1 06 g Data analysis mode Figure 3 33 Rack detection is successfully completed 3 6 Calibration of the instrument settings In flow cytometry fluorescence intensity is used to distinguish between positive and negative populations of particles The reproducibility and stability of the fluorescence signal over time is of vital importance In order to ensure a stable measurement that is independent of time and the specific analyzer instrument calibration is performed Fluorescence calibration curves are calculated by using standardized fluorescence microbeads that have predefined sizes and fluorescence intensities A linear regression equation is calculated from the instruments response in mean or modal histogram channel values to these predefined values 3 6 1 Performing a fully automated calibration 1 Prior to beginning calibration ensure that the single tube holder is correctly attached Figure 3 34 The single tube holder and orange acquisition button 2 Activate the reader by clicking on the Barcode icon and present a vial of MACSQuant Calibration Beads in front of the barcode reader To proceed with the calibration process select Yes 56 3 Follow the dialog box instructions i e place an empty tube into t
162. n off the Multilayer mode Switch on Multilayer mode to view all regions on a plot Switch off the Multilayer mode to only view the region displayed on the plot header e Dot plot Density plot 9 Axis Anti IL 17 PE versus Anti CD4 FITC A rectangle region was drawn P3 using the rectangle tool to select for IL 162 17 CD4 viable lymphocytes MiN2009 02 27_2053 021 P14 P2 CD4 FITC A le 2 le 1 1e0 lel 1e2 1e3 Anti IL 17 PE A Figure 6 67 A rectangle region P1 P2 P3 was drawn to select for activated IL 1 7 CD4 lymphocytes 10 Axis Anti IL 17 PE versus Anti CD154 APC The region P3 gate i e P1 P2 P3 was displayed using the axis Anti IL 17 PE versus CD154 APC MiN2009 02 27_2053 005 P1 P2 P3 f live j y MiN2009 02 27_2053 005 Pl P2 A final population of enriched activated IL 17 secreting CD4 T cells are shown MiN2009 02 27_2053 021 P1 P2 P3 le3 BT POPAULS PRPAPRURS CD154 APC A 1e2 1e3 1e0 lel Anti IL 17 PE A Figure 6 68 The region P1 P2 P3 was displayed using the axis Anti IL 17 PE versus CD154 APC 11 Click to expand the gating strategy in the Samples menu i e Mi 2009 02 27 2053 005 100 0 e2daa4 163 IL 17 Analysis_complete mq admin File Edit View Mode Analysis Window Help aaa dododd daek air Owa Samples Experiment Tools Channels pipz 181 T PINP24P3 1 81 T a PAPRLA PI PAPSURE Figure 6 69 An
163. n process Note When scanning MACS Reagents the MACSQuantify Software will prompt the user to place the vial s on the MACS Reagent Rack Note Contact your administrator if the code reader fails to recognize a reagent vial Note Administrators and Custom Users should refer to section 6 6 for further assistance 5 9 Printing in Express mode The MACSQuantify Software uses installed windows printer drivers to print active workspaces Note The HP Universal Print driver has been installed on the MACSQuant Analyzer and has been tested with the following printers Hp Laserjet P2055d P3005n CP1515n PC2025n Hp Officejet Pro 8000 For a complete list of printers compatible with the HP Universal Print driver please visit www hp com go upd Please note the only the above mentioned printers have been tested with the MACSQuant Analyzer Note It is also possible to print to a network printer Please contact your MACSQuant Analyzer administrator or Miltenyi Biotec technical support for more information To print active workspaces 1 Open the desired workspace or analysis window 90 2 ba S j 3 Select the desired printer Click Print The printer can be networked to or directly connected to the MACSQuant Analyzer or to the PC running the MACSQuantify Software 4 The active workspace is printed as shown below Date 2005 10 16 Version 2 0 ALA orl ts rend meron at Bora el A PE GSA i Te 1
164. nditions of operation 15 30 C with 0 85 humidity at a maximum altitude of 2000m The MACS MiniSampler is not specified for use in the cold room 203 The MACS MiniSampler has been investigated by Underwriters Laboratories in accordance with the standards UL 61010 1 and CAN CSA C22 2 No 61010 1 and meets the intent of the directive 2004 108 EC electromagnetic compatibility Compliance was demonstrated by conformance to the following harmonized European Standards which have been listed in the Official Journal of the European Communities EMC EN 61326 1 EN 61000 3 2 A C US EN 61000 3 3 Compliance was demonstrated by conformance to the following FCC Rules of the Code of Federal Regulations 47 CFR 15 class B 204 13 Technical service Miltenyi Biotec offers a full range of customer technical support options for your MACSQuant Analyzer For support and technical questions or if you think your MACSQuant Analyzer is malfunctioning please contact your local Miltenyi Biotec representative or Miltenyi Biotec s technical support team Germany Austria Switzerland Miltenyi Biotec GmbH Friedrich Ebert Str 68 51429 Bergisch Gladbach Germany Phone 49 2204 8306 830 Fax 49 2204 8306 89 macsTec miltenyibiotec de USA Canada Miltenyi Biotec Inc 12740 Earhart Avenue Auburn CA 95602 USA Phone 1 530 888 8871 Fax 1 530 888 8925 Toll free 800 FOR MACS Toll free 1 800 367 6227 macs miltenyibiotec com
165. needle arm into the sheath flow port 47 5 Click Save calibration File Edit View Mode Analysis Window Help S a e z a 34 39 Gi E m ul puin omne lIBE Rist Uptake unit calibration 1 First push the Start calibration button to begin the calibration be careful the hardware moves Show backup registry 2 Move the needle and sampler by the hand to the specified position and press Save calibration 3 Test your calibration using the Test button select position you want to test F Calibrate uptake unit Calibrate rack detection Update software Import Export user settings Remove extemal media inane dt L__stercaibraion _ __Save caitraion _ MACS Quant live support L_sterceibraton J Seve caitraton _ Test _Stercatbraton J Save calibration _ _stetcetiraion J __Seveceltraion J Tet wT Ne a NN CVO p p a Se ee eee Zoftset 0 Start calibration Save calibration N A FANTAS S NG NP NS 60 45 00 M Figure 3 19 Coordinates of the needle arm are saved 6 The closed green circle indicates that calibration of the needle arm position is completed 7 Click Test to confirm that the correct coordinates are saved Calibration of the single tube rack 1 Gently insert the single tube rack it into the corresponding slots located at the front of the instrument position 5
166. ng fluids out of the tubing i e all fluids inside the tubing should be empty Remove the screws that attach the pump syringe safety guard and then remove the safety guard covering itself by lifting it Loosen the plunger lock screw by turning it counter clockwise approximately three full turns Lower the plunger lock assembly by firmly pressing the plunger lock screw down 182 6 Unscrew the syringe from the dilutor housing by turning it clockwise 7 To install the newly cleaned syringe carefully insert the syringe into the dilutor housing and screw counter clockwise until resistance can be felt Note To clean the syringe carefully remove the plunger from the syringe Remove salt crusts with distilled or deionized water Use distilled or deionized water to wet the plunger and carefully push the plunger back into the syringe Dry the plunger lock screw before proceeding with installation of the syringe 8 Push the plunger holder assembly up to the syringe plunger and tighten the plunger lock screw 9 Turn onthe MQ 10 Prime the MACSQuant Analyzer 9 1 2 Exchanging fluid containers When one or more containers need replacing follow the instructions below to ensure a safe exchange Only exchange one container at a time and note the corresponding color coding of the container to be exchanged 1 Unscrew the bottle closure counter clockwise and remove Close the container with a suitable blue cap and then remove
167. niversal Print driver has been installed on the MACSQuant Analyzer and has been tested with the following printers Hp Laserjet P2055d P3005n CP1515n PC2025n Hp Officejet Pro 8000 For a complete list of printers compatible with the HP Universal Print driver please visit www hp com go upd Please note the only the above mentioned printers have been tested with the MACSQuant Analyzer Note It is also possible to print to a network printer Please contact your MACSQuant Analyzer administrator or Miltenyi Biotec technical support for more information To print active workspaces 1 Open the desired workspace or analysis window 2 Click 113 3 Select the desired printer Click Print The printer can be networked to or directly connected to the MACSQuant Analyzer or to the PC running the MACSQuantify Software The active workspace is printed as shown below Date 20006 23 Version 1 2 0622 R pre ig Dii peal Time i i E a eT eT eT T 1 ed mi _ DO Fi 10 Te TT Se T Tat ed Vindiiue A Pats PEA MME 12 10_ 2083 01 pew Legend laeor Popdebon Count Fl igre can TE PTF imbis ekp 11176 PTFS Louies ec Fas Pee CON Nirai Frail Tis TT oe eT Tat 7 PEA aksi m ird i E616 pow 15 Results pos 16 lag 1 ot Freq Conc imi dual Viable leukocytes wer 82 5 2244005 E iai Viable CO14 momocytes wer 9 7 1 56E 005 W Ts 17 oT oe Winbond Page 11 Figure 6 13 Half size example print o
168. nstrument on overnight or over the weekend Configuring automatic shutdown Note Configuration of the automatic shutdown can only be performed by Custom users and administrators 179 1 Click Edit Options Software and Timers Options E Users Experiment E Instrument Software Keyboard Standby timer 120 min Acquire 5 min Shutdown timer Export Needle priming time O min Backup H Regions Views Timers Shutdown default behavior Analyse mode Instrument off Figure 7 1 Timers window 2 Click on the Shutdown time field Modify the time minutes using the slider bar or keyboard 3 Click Apply and OK 180 8 MACSQuant Live support MACSQuant Live support is a real time diagnostic service provided by Miltenyi Biotec technical support Highly trained MACSQuant Experts can be reached in real time to assist with any queries you may have Note As an option is it possible to use a web cam in communication with MACSQuant technical support If no web cam is provided please contact your nearest MACSQuant Specialist Note The MACSQuant Analyzer must have network access to the internet for live support Contact your local IT administrator if this is not the case To receive remote assistance 1 Select the Tools menu 2 Click MACSQuant live support A popup HTML field will appear Complete the fields with your information and detail any querie
169. o1i Instrument settings Channel Compensation Matrix v FL7 0 0 1 0e1 0 0 0 0 FITC A 0 0 0 0 0 0 1 0e 1 1 0 1 0e2 1 0e1 1 0e0 1 0e1 1 0e2 1 0e3 PE A Figure 3 45 Compensated density plot with corresponding compensation matrix Population A bright PE stained cells is no longer detected as a relatively strong fluorescence signal in the FITC channel CH2 and has therefore been successfully compensated 3 7 2 7 color compensation In combination with the grouping function it is possible to perform auto compensation with 7 stains fluorochromes 66 To perform 7 color compensation Prepare single stained cells 1 2 3 4 5 6 7 Determine the cell number of a PBMC sample Centrifuge cells 300xg for 10 minutes Aspirate supernatant and resuspend cells in sample buffer at a concentration of 1x107 cells per 100 uL buffer Aliquot to 9 tubes Add an appropriate fluorochrome conjugated antibody at the recommended titer to a single tube Repeat process for another six tubes using suitable fluorochrome conjugated antibodies i e a total of 7 tubes containing single stained cells Note For example the following fluorochromes may be used for single staining cells VioBlue FITC PE PE Cy5 PE Cy7 APC APC Cy7 Leave one tube as an unstained blank Add propidium iodide Pl to the final tube at the recommended concentration see corresponding datasheet Mix well and
170. on Beads abg 5mL EERE Se Ea EA Ea a E ES gt SAG eal eae Met Sd EE recep he Ba fee ag 01 via 5 vja i See section 6 6 for information about reagent management Settings tab 11 Select either an Express or Custom mode of analysis Annotations Autolabel Settings Annotations Autolabel Settings Custom Express Custom Express C Instrument setting Type Analysis v C Analysis template Mode v C Gate ClEvents 10 000 LJ For Custom mode analysis click the checkbox associated with the Instrument settings Analysis template Live gate and Events options to activate these features Instrument setting previously saved instrument calibration and compensation settings can be loaded and applied by checking the adjacent box Analysis template previously saved cell analysis templates can be loaded and applied by checking the adjacent box Live gate check the adjacent box to activate live gating a live gating strategy can be saved as an analysis template for future use Events check the adjacent box to stop data acquisition after a defined number of events is obtained in this example 10 000 events Note It is recommended to limit measurement by volume Volumetric measurements allow for absolute cell counting 12 Ensure that reagents samples and buffers are correctly positioned Check that the waste bottle is empty Note
171. oncerning gating or defining regions of interest refer to section 2 2 8 151 6 12 1 Creating a new analysis template or analysis window Analysis templates consist of a plot template new analysis window and a gating strategy Analysis templates can be created post acquisition or during acquisition in Live mode To create a useful analysis template whether in Live mode or post acquisition the Experiment settings must have been correctly defined See section 6 3 6 for more details An analysis window can be saved as an analysis template Click the New analysis window icon Eh or use the file menu option Window and New analysis window to create a new analysis window To create a new plot window 1 Click the icon or use the file menu option Window and New analysis window Create New Plot Window Figure 6 55 Available analysis templates are displayed by the Create New Plot Window 2 Click on the required analysis template 3 The analysis template will open as shown below Pie Edt View Mode Analyses Window Hep aalas ICEErRRRE Eaa E maaa o admin Note Multiple analysis windows can be opened These can be of single or multiple experiments 152 Note If multiple analysis windows are open use the top menu bar icons to display the previous and next analysis window 6 12 2 Choosing a display format for plots and histograms The layout of an analysis template is predefined as follows
172. ontrol applications optimized evaluation of MACS Cell Separations as well as standard immunofluorescence analyses The system includes the MACS Cell Enrichment Unit that is crucial for the reliable detection and analysis of rare cells it is directly controlled by the MACSQuant Analysis Software to enable the fully automated processing of samples and cell analysis Automation can further be extended to multisample processing when combined with the MACS MiniSampler for convenient hands free operation Figure 2 7 Front image of the MACSQuant Analyzer the access cover was made transparent for the purpose of illustration The MACSQuant Analyzer e Compact benchtop design e Straightforward multiparameter cell analysis from simple cell counting to sophisticated flow analysis e Absolute cell counting volumetric e Highly sensitive detection of rare cells e Fully automated multisample labeling and analysis 31 3 Assembly and installation of hardware The following section describes how the MACSQuant Analyzer is unpacked and installed for first use 3 1 Unpacking the MACSQuant Analyzer Read through the following instructions carefully before commencing the installation procedure Before opening the transportation box check for any visible external damage to the box Check also to see if the shock and position indicators if present suggest incorrect transportation of the instrument If there is apparent damage please contact te
173. ormation Ensure that The samples are correctly positioned on the reagent rack and that the MACSQuant Analyzer is provided with adequate buffer The waste bottle is empty The instrument is correctly calibrated and compensated Click Start Measurement CJ to start analysis mek O Custom Logout Help mq Express User S BIS im amp Definition Rack Chill 5 tube rack Filename EU2009 09 28 001 mqd Sample ID CD_14 Cocktail Description PMBC Preparation Mode Analysis Figure 5 17 The experiment has been defined By clicking Start Measurement the instrument will change to Acquisition mode 88 Note By saving the Experiment definition see Step 7 above the user can reapply the definition by clicking selecting the appropriate file and clicking Open 10 The instrument will proceed to Acquisition mode 11 Following data acquisition the MACSQuant Analyzer will automatically proceed to Analysis mode mq Express User u olola MM Bs ve coon tome ch Ww 1 KLa2008 07 17 016 1 KLa2008 07 17 016 en 1000 1000 Definition Acquisition 750 70 a 100 00 T Analysis ss lt q lt Q 500 g 500 on on Rack Single tube rack v 250 250 Filename EU2009 09 29 001 mad he 5 SN 1 02 1 01 10 11 12 13 0 250 500 750 1000 1 KLa2008 07 17 016 1 KLa2008 07 17 016 Mode Analysis template v CD14 v PI A 1 02
174. ots in a plot layout is given by a number from 1 to 16 The position of a plot in the plot layout is assigned by letters abcdefg etc where position A is at the top left hand corner of the plot window and the last letter used is at the bottom right hand corner of the plot window Letters can also be reused e g aabb this allows the user to assign a plot type over two or more positions For example Plot4 can be described as 4 PPPP abcd o 4 p o j g That is 4 plots will be assigned to the total workspace Each plot will be dot plots P abcd denotes that each dotplot will be placed in each corner of the workspace Another example Plot3 can be described as 3 PPS abcc That is 3 plots will be assigned in total Of which two will be dot plots PP and one will be a Statistical table S The dot plots will occupy the upper left and upper right corners of the workspace ab The Statistics table will occupy the entire lower half of the workspace cc Note Hyphens are used to separate the conditions used to describe plot layouts e g 3 PPS abcc Only hyphens may be used 4 Configure the dot plots as necessary Click Apply to implement changes Click OK to close the window To assign default properties for displayed plots histograms and tables Views 1 Click Edit Options Software and Views Options E Instrument Software K
175. ould be changed e g E Note Regions are assigned numbered in ascending order i e the first region to be drawn is assigned the value 1 the second is assigned the value 2 etc 3 Select a new color Click OK 4 Click Apply to implement changes Click OK close the window To assign default properties for Windows and Window Templates 1 Click Edit Options Software Windows Options H Users Experiment E Instrument i Software Delete Always ask Keyboard Timers Acquire Export Backup Regions Colors Templates Views Windows Figure 6 53 Changing the default color settings for windows 2 It is possible to configure a warning prompt when users click in order to close an analysis window e Never ask the window will immediately close when is clicked 148 e Always ask the user will be prompted to Close the current window when gt is clicked 3 Click templates to configure the layout of window templates Options Users Experiment H Instrument B Software Rows 2 v a Cos 3 a Keyboard i l j Timers Acquire E Export E 3 PPS abcc up i 4 PPPP abed ji he 4t PPPS abedddddd Windows 6 PSPSPS abbeddeff 6a PPPPPP aabaacdef Vi 9 PPPPPPPPP abedetghi Views Templates Plots types are assigned the following nomenclature P Dot plot D Density plot H Histogram T Text S Statistic N None blank The total number of pl
176. out the necessity of long flow analysis times The rare cell must be labeled with one of the MACS Microbead reagents as well as the fluorochromes of interest This process in the presence of the MACS Enrichment Unit provide a flexible tool to pre enrich a particular cell type prior to analysis Note These cells cannot be retrieved Please refer to section 9 1 3 for more illustrated instructions on exchanging the MACSQuant Column Remove the column substitute and install the MACSQuant Column according to the following instructions 1 Open the front door and note the position of the tubing and the pre installed substitute dummy of the MACSQuant Column 40 2 Using both hands hold the top and bottom of the column substitute and pull gently but firmly to remove it from its slot in the MACS Enrichment Unit 3 Place a paper towel under the column substitute Hold the column substitute in one hand and gently unscrew the upper column connector anti clockwise Tilt the column substitute downwards to empty any fluid Then unscrew the bottom column connector Store the column substitute for later use 4 Insert one end of the new MACSQuant Column into the bottom column connector and gently screw in the column by turning it clockwise until you feel resistance Point the column towards the top of the device and screw in the top column connector Note The column has an appropriate orientation The top portion of the column has a 3 mm
177. ovide information on physical characteristics such as size and granularity Also the laser light excites the fluorochromes on fluorescencely labeled cells the light emitted from each excited fluorochrome is measured by color detectors after passing through the respective filters fluorescence channels Finally the cells are discarded into the waste container 2 2 7 Displaying flow cytometric data Flow cytometry data can be displayed in five different formats by the MACSQuantify Software dot plot histogram density plot and statistic Each category is briefly discussed below however it is worth noting that data are normally visualized as one parameter histograms or two parameter dot plots For more information on using the MACSQuantify Software for data analysis and displaying charts refer to section 6 12 Dot plot A dot plot may also be referred to as bivariant display scattergram or in some cases bitmap In this form of analysis each cell event is represented as a single dot on a two axis scale chart The position of the dot on the x y scale is dependent on the intensities of the measured parameters for that cell event Characterization of a cell population is typically achieved by displaying a dot plot where side scatter SSC y axis is plotted against forward scatter FSC x axis 1000 act Granulocytes _ fa 500 ESC A Monocytes M Too 0 rl 500 foo 1000 FSC A Lymphocytes Figure 2 1 A
178. owed by Deselect All Single right click on Selection deselection of an entire sample column header r VatawTs column Select Used in Col Deselect Col Clear Col Clear Selected in Col N I I j NA QS NOF i IEN a a WO ri j Single right click on Selection deselection of an entire sample 1 2 3 row A row In this example Row A is selected for sample labeling and measuring Select Row Select Used in Row piy p gamn ga i Deselect Row Tanks hee Row B is selected for sample measurement Clear Row i N s Na Na Clear Selected in Row O n ly PTN EN r a GON ae EON mm EE Na Single right click over Right click over a single rack position to a single rack position completely clear this position In this example position A2 will be cleared Grouping function Only rack positions that are adjacent and in columns can be grouped To group several adjacent rack positions 1 Select the rack positions J 2 Click Group See 6 8 3 grouping sample for more information Cear Group Table 6 7 An overview of the possible configurations for rack positions 121 To configure a sample rack 1 Click on a sample position s using the left mouse button or touchscreen An entire row column or table can be selected using see Table 6 7 for more details 2 Use left mouse button or touchscreen to toggle between O and a designed sample will be measured and associated
179. per valves Figure 12 3 The hinged washing station cover is opened to the left and gives access to the washing station barcode readers the peristaltic pump and the tubing of the washing station Both hinged covers can be removed by opening the door and lifting the covers vertically off of the guides The bottom cover Figure 12 1 gives access to the lower valves and can be removed by pulling gently toward you Fluidics system The MACSQuant Analyzer fluidics system supplies the instrument with all buffers and solutions required during and after operation The fluidics system Figure 12 3 comprises of the tubing with color coded markers to assist correct connection to the respective fluid container as well as their guides three solenoid valves three valves a 196 flow cell four pressure sensors two syringe pumps and the MACSQuant Column Figure 12 3 MACSQuant Column The MACSQuant Analyzer Column Figure 12 3 is an optional component that is designed for the enrichment of cells using MACS Technology before fluorescence cell analysis Up to 5x107 cells from up to 5ml can be enriched in one isolation using the column the column should be replaced every three months For further details refer to the respective product data sheet Fluid containers and fluid container holders Two orange baskets Figure 12 1 holding two fluid containers each are located on either side of the instrument The containers supply the MACSQuant Analyzer
180. pter Important information section 1 The MACSQuant Analyzer is a bench top instrument that fits neatly onto a benchtop and into standard sized laminar flow or safety cabinets It should be installed ona stable flat and vibration free surface The operating environment should be dust free sufficiently ventilated and free from sources of electromagnetic radiation In order to ensure a flat surface in laminar flow hoods the MACSQuant Analyzer can be placed on a MACS Laminar Hood Plate cat 130 093 246 34 Note Before operating the MACSQuant Analyzer for the first time carefully read the user manual and contact your local Miltenyi Biotec representative for assistance Note When delivered the MACSQuant Analyzer fluidics system is delivered dry i e without storage solution 3 2 1 Connecting the fluid containers and fluid sensor cables Operating the MACSQuant Analyzer requires running buffer washing solution and storage solution Always operate the instrument with ready to use MACS Buffers and solutions The MACSQuant Analyzer is delivered with four empty fluid containers bottles which can be found in the orange fluid container baskets connected to the instrument The bottle closures consist of a fluid uptake port for solutions green blue and black closures or a fluid outlet port waste container as well as an electrolyte sensor for measuring liquid levels The fluid containers bottle closures and fluid sensor cables a
181. r MACSQuant CAN Bus DC Analyzer Output labeled External CAN 100 240 VAC AC Output labeled 50 60 HZ Bottle Sensor CAN Bus labeled CAN1 or CAN2 Specification Pins 1 4 8 NC Pin 2 CAN L Pins 3 6 GND Pins 5 9 24 VDC 2A Pin 7 CAN H Pins 1 2 3 4 5 5 VAC 10 kQ Pins 6 7 8 14 15 GND Pins 9 10 11 12 13 Input Pins 1 4 5 8 9 NC Pin 2 CAN L Pins 3 6 GND Pin 7 CAN H 200 Parameter Model RS232 Interface labeled COM 1 RS232 Interface labeled RS232 AUX Not in use RS232 Interface DC Output labeled RS232 BCR Specification MACSQuant Analyzer 2 x 5AT 250V Pin 1 DCD Pin 2 RxD Pin 3 TxD Pin 4 DTR Pin 5 GND Pin 6 DSR Pin 7 RTS Pin 8 CTS Pin 9 RI Pins 1 4 6 7 8 9 NC Pin 2 RXD Pin 3 TXD Pin 5 GND Pins 4 6 NC Pin 1 Input Pin 2 RXD Pin 3 TXD Pin 5 GND Pins 7 8 Shorted Pin 9 5 VDC 0 5 A Parameter CAN Bus DC Output labeled External CAN USB port labeled USB Ethernet port labeled Ethernet VGA Interface labeled VGA Specification Pins 1 4 8 NC Pin 2 CAN L Pins 3 6 GND Pins 5 9 24 VDC 2A Pin 7 CAN H Pin 1 USB1_ 5V Pin 2 USB1 Pin 3 USB1 Pin 4 USB1_GND Pin 5 USB2_ 5V Pin 6 USB2 Pin 7 USB2 Pin 8 USB2_GND Pins 4 5 7 8 NC Pin 1 TXD Pin 2 TXD Pin 3 RXD Pin 6 RX
182. r instructs the user how to install the MACSQuantify Software onto an independent personal computer and onto the MACSQuant Analyzer 4 1 Installing software onto a personal computer 1 2 3 4 Note The recommended PC specifications to run the MACSQuantify Software follows Operating system Microsoft Windows XP SP2 is a minimum requirement although SP3 is preferred Memory 1GB minimum Insert the MACSQuantify Software DVD into the computer DVD drive The installation program should automatically run If this is not the case using Windows Explorer navigate to the root directory of the CD ROM drive and execute the file installICAP bat At the prompt Do you want to install a new cap package Y es A bort Select Y to continue with the installation or A to abort the installation At the prompt Install on MACSQuant Y es N o A bort Select N when installing the software onto a PC Select A to abort the installation At the prompt Do you want to keep old configurations and settings Y es N o A bort Select Y when current software configurations and settings should NOT be deleted by the new installation Select N when current software configurations and settings should be deleted by the new installation Select A to abort the installation 70 5 At the prompt Do you want to keep all data files Y es N o A bort Select Y when saved data files should NOT be deleted by th
183. rd scatter vs side scatter using linear scales B Histogram showing fluorescence intensity linear scale vs cell count C Histogram showing fluorescence intensity log5 scale vs cell count D Histogram showing fluorescence intensity hlog scale vs cell count There are occasions when the difference between fluorescence values in a dataset are relatively small for example when using fluorescent probes to measure quantitative changes in cellular DNA during cell cycle A linear scale must therefore be used in order to visualize these subtle changes see Figure 2 5 cell cycle OO1 P1 call ryrie 1014P2 i 3510 i 300 a00 3030 ro 2500 600 500 2000 400 1530 300 200 100 Lt 1030 SJU 0 500 730 100C 1 0e 2 1 3e1 10e0 10e 1 0e2 1 0e3 PLA PLA 0 Figure 2 5 Measuring quantitative changes to cellular DNA during cell cycle DNA was labeled using the fluorescent probe propidum iodide PI A linear scale left and log5 scale right was used to plot fluorescence intensity against cell count Subtle changes to the quantity of DNA can only be visualized using a linear scale left As a general rule however changes in fluorescence intensity usually span over several orders of magnitude and therefore a logarithmic or biexponential hLog scale should be used 27 2 2 8 Analyzing flow cytometric data using regions or gating It is usually necessary to analyze cell subpopulations or at the very le
184. re color coded for easier handling see Table 3 2 Container Container Running Storage solution Buffer black blue Washing solution green Table 3 2 Symbols and color coding of fluid containers 1 Install one fluid at a time Place a new bottle into the orange fluid container basket Please note the corresponding color coding see Table 3 2 2 Unscrew the lid of the bottle and replace it with the appropriate bottle closures Do not unscrew the fluid container lids until the bottle is placed in the basket 35 3 Remove the fluid sensor cables and bottle closures from the packaging Blue green and _ Red colored All bottle tubes black colored closure waste packed in a single closures and is packed ina bag accompanying Separate bag hydrophobic filters in a single bag Figure 3 5 Packaging format of the tubing bottle closures caps and hydrophobic filters 4 Remove the end caps from the bottle distribution block Figure 3 6 Before inserting the tubing into the fluid ports it is necessary to remove the end caps from the bottle distribution block 5 Connect the tubing to the appropriate color coded fluid port on the back of the MACSQuant Analyzer Figure 3 7 Connecting the tubing to the bottle distribution block 36 6 Attach the sensor cable plug to the socket for sensor cables at the back of the MACSQuant Analyzer and fasten securely Figure 3 8 Connecting the sensor cables
185. re performing a backup the user is prompted to verify which file types are to be backed up b always all files all files are always backed up there is no user prompt to verify this procedure C always data files only data files are backed up there is no user prompt to verify this procedure 3 Files Deletion a automatic automatically overwrite or delete files during backup b ask the user is prompted to verify deletion or overwriting of files during backup 4 Click Apply to implement changes Click OK to close the window 6 10 5 Configuring network settings administrators only In order to have remote assistance and data backup to a network location it is necessary to configure the MACSQuant Analyzer network configuration 137 To configure the MACSQuant Analyzer for network access Note It will be necessary to contact your local network administrator may also be necessary to seek assistance from your Miltenyi Biotec technical support 1 Click Edit Options Network setup 2 Consult your IT administrator in order to configure the following settings Options Users Experiment E Instrument reg Network setup Connection Software Network MAC address Hostname Domain suffix 3 Expand the tree Network setup Meteiksetue I and configure the following settings Options H Users Experiment E Instrument Network setup DHCP IP address IP addre
186. red in user designated folders for later analysis These folders are assigned for each user as either private or public access by the MACSQuant Analyzer administrator The MACSQuant Analyzer can also optionally perform pre enrichment of magnetically labeled cells before flow cytometric analysis This feature is based on the renowned 20 MACS Technology and is particularly useful for the analysis of rare cells labeled with MACS MicroBeads Operation of the instrument is extremely simple through the use of the TFT color touch screen and intuitive screen menus built into the MACSQuant Software The user has the option of performing simple analyses pre programmed into the software using the Express mode or of customizing sample analysis protocols and automation programs using the Custom mode Data analysis using a variety of display options and functions can be performed on the instrument and the software has been configured for user friendliness and to provide highly flexible functionality Finally standard MACSQuant Analyzer Buffers and Solutions which are directly attached to the instrument are sterile ready to use and designed for optimal instrument performance The instrument also provides a color coded LED warning system which illuminates the fluid containers to inform the user when buffers need to be exchanged or waste removed 2 2 Applications The MACSQuant Analyzer is more than just a flow cytometer The instrument was design
187. rify this procedure C always data files only data files are backed up there is no user prompt to verify this procedure 3 Files Deletion d automatic automatically overwrite or delete files during backup e ask the user is prompted to verify deletion or overwriting of files during backup 4 Click Apply to implement changes Click OK to close the window To assign default drag and drop properties for Regions 1 Click Edit Options Software and Regions Options Users Experiment Instrument Software Keyboard Drag amp drop Regions Timers Create always link Acquire Export Change always ask Backup 3 Colors E Views Figure 6 51 Changing the default settings for drag amp drop of regions 147 2 Use the dropdown lists to activate deactivate a feature Create when creating a new region Change when changing a region 3 Click Apply to implement changes Click OK close the window To assign default color properties for Regions 1 Click Edit Options Software Regions and Colors Options H Users Experiment E Instrument Software 1 mm black 6 magenta Keyboard Timers darkgreen Region colors Acquire H Export 8 Ml darkred Backup Regions 9 E darkblue Windows 10 E darkcyan Templates 4 Views Cancel Figure 6 52 Changing the default color settings for regions 2 Click on the color panel button adjacent to the region that sh
188. rlay tab A script was used to create the following text table srv2008 12 10_2063 015_pos Edit text via Properties Apply Legend Region Population Count P1 Debris esclusioni 13552 P14 P2 Viable leukocytes 11178 PI P24 P3 Granulocyte exclusion or PI P24 P34 P4 CD14 Monocytes 790 Scripts are provided by MACSQuant Analyzer specialist Please contact your Miltenyi Biotec advisor for further information E Properties Overlay Overlay Population If a MACSQuantify Script has been used Text tab it possible to add or remove regions ion stv2008 12 10_2063 015 pos P1 m f jon 2 srv2008 12 10_2063 015 pos P1 P2 m populations by using the Population check jon 3 srv2008 12 10_2063 015 pos P1 P25P3 E box In the following example all four regions ion 4 520081210_2068 015 poss APPA E P1 P2 P3 P4 of a gating strategy were checked and therefore displayed sr 2008 12 10_2063 015 pos Z Population stv2008 12 10_2063 015 pos P1 m Legend Z Population stv2008 12 10_2063 015 pos P1 P2 m Region Population 1 2 Z Population 3 srv2009 12 10_2063 015 pos P1 P2 P3 m PI 4 PI P2 stv2008 12 10_2063 015 pos P34P4 m AER v Population Table 6 11 An overview of the Properties settings for Text boxes 6 13 Working with regions or gates For background information conc
189. s 6 10 4 and 6 10 5 of this help guide Note Before performing Backup ensure that the desired backup media is accessible to the MACSQuantify Software Backup media The backup procedure QD searches for backup media in the following order 1 A designated folder located on a local area network this must be setup by an administrator with assistance from Miltenyi Biotec technical support 2 Amemory stick attached to the USB port on the MACSQuant Analyzer 3 Arewritable DVD 4 If none of the above are found the MACSQuantify Software reports an error No valid backup devices found 133 6 10 1 To perform a backup to a rewritable DVD 1 2 3 4 5 6 7 8 Ensure no USB stick is installed and that no network drive has been defined as the default location for backup files Please contact your administrator for further advice Insert a rewritable DVD into the MACSQuant Analyzer DVD drive Only DVD R or DVD RW media may be used DVD RW and CD media types are not currently supported Wait for 10 20 seconds after inserting the DVD into the drive Click the backup icon located on the top menu bar a The files will be written to DVD Note Depending on the amount of data the backup procedure may take several minutes When the progress bar displays 100 the MACSQuantify Software will verify the data once again this may take a few minutes to complete Note At this stage data will NOT be deleted from
190. s and the MACS Reagent RACK 4ucsiccccscenenenereceees 39 3 2 5 Switching ON OFF the MACSQuant ANAIV ZOD visucacecencneccenenecsenenscsesenscecsenenssesenees 40 3 2 6 Installation of the MACSQuant Column optional visicecccucecenenenenensesecenerenenenensnees 40 S22 i HIS tAIAUON OF the WED CAIN espeare an EE R a A widest A ia 4 FAO MIS CAI AUIOIN CHEEKS C rirse na ipa RE A E ET ee ea ee oia cae ese ae 42 3 3 MATERIALS REQUIRED FOR OPERATION OF THE MACSQUANT ANALYZER ccccceeeceeeeuceeeceeeueueeeneas 43 3 3 B ffets And Solutions roerei desta cau huine EEEE AE EE utopian nau tis EEE EEEE EA T A A E GEER EAA 43 3 3 2 Hardware and CISDOSADIOS uisicacenccuccenencccenenscsusensnsesersnsesesensasenenensetensesesensneesensnees 44 3 4 MATERIALS REQUIRED FOR MAINTENANCE OF THE MACSQUANT ANALYZER seeceeeeeeeeeeeeeueeeeeeees 45 PAA OMI LOND Seaside r aha an clot E he id debe ais dinaelic tata A 45 Bol FLAW AIC AAEE rit ch ate id tian cleat ecto an A OEE E E A E NE 45 3 5 CALIBRATION OF THE MACSQUANT ANALYZER HARDWARE ccceeeceeceeeeeeeeeeeeeeeeeueeeusueeeneueeenees 45 3 5 1 Calibration Of the uptake UNM ti isicccccccccececececensnsccceeesesereneneneneaeesacetetenenensnsnsasssess 46 3 5 2 Calibration TACK detection ives cscs suite teautie wad povesdseddoredsiedbuanidesnidvelabaarscccdentuensbedeses 54 3 6 CALIBRATION OF THE INSTRUMENT SETTINGS cccecccceeveceeeaceevacueeaeeaeateaueneaneavaneavenvausevaeenvanses 56 3 6 1 Performing a fully autom
191. s you may have using the Message Question box E HTML Help To talk with a representative please answer the following First name Doe Required Last name Bloggs Required Country City USA Required Email address joe bloggs laboratory com Required Phone number Country region Number with area city code 1 99999999999 Message Question I would like assistence with 3 Click Submit 4 Live support will commence 181 9 Maintenance 9 1 General maintenance The following section describes procedures required for maintenance of the instrument 9 1 1 Pump maintenance The syringe pump requires periodic maintenance It is recommended to clean the dilutor housing and the syringe plunger every three months Removing salt deposits as they appear can prevent leakage of the tubing system The pump seals or the syringe should be replaced once a year Depending on the level of use and general instrument maintenance however these parts might need to be exchanged more frequently To exchange the pump syringe 1 2 3 4 5 Make sure the fluidics system is shutdown Switch OFF the power and unplug the MACSQuant Analyzer from the power outlet Open the front access cover the syringe pump plunger should be at its top position Unscrew the bottle closures blue green black and hold the bottle closures below the height of the dilutor valve gravity will draw any remaini
192. sing the 6 i 7 Properties option from the dropdown menu 1 Click the icon located adjacent to the Statistic textbox 2 Click Properties an The properties window will appear Note Refer to Table 6 10 below for an overview of the various display Properties that may be modified for the Statistic option using MACSQuantify Software To change the contents of the statistic text box refer to Error Reference source not found below 158 Tab Category Property Associated screenshot Description option View Options Header eade Check the Header box to include this eatus enera information in the text box Table Check the Table box to include this information in the text box Column headers titles of the table can be displayed in two formats shown using annotations and shown using channel names Annotations are defined by the user For example in example Error Reference source not found channel FL1 A was annotated as VioBlue A Region Regions The settings for regions and functions are functions identical to those for dot plots Feature Features o meeen ai The recorded time scatter and fluorescence function a data acquired from each channel may be cesta aT displayed using the Features setting The data KHANA a shown will be dependent on the selected Xsareare regions see Region functions Features can be XRT AAPCOVTA displayed cw or hidden tas by selecting or deselecting a fe
193. sions when the sample size is of course greater aliquots of the sample must therefore be spanned over two or more tubes By grouping these samples the acquired data will be consolidated into a single file on the hard drive which can also be analyzed in a single data file or analysis plot This can be easily accomplished by grouping sample prior to data acquisition Refer to section 6 8 3 for more details If grouping was not performed prior to data acquisition it is still possible to group samples post acquisition A D all plots i _Sample grouped into B grouping a single file Figure 6 77 Schematic of the grouping process To group samples 1 Click File Open and navigate to the files that must be grouped Highlight the files and click Open Data files adm2009 09 01_2022 001 adm2009 09 01_2022 002 adm2009 09 01_2022 003 g m o i o i eo o w 0 oO a a E MC_CD4_T_Cell_Cocktail_h E MC_CD3_Pan_T_Cell_Cocktailh E MC_CD19_B_Cell_Cocktail_h O MC_CD14_Monocyte_Cocktail_h ag IL 1 9 CD34cocktail hlog 49 CD34_CD133 cocktail log5 g 3 g E m m 3 a 49 CD34_CD133 cocktail hlog E CD34 Stemcell log5 C 2009 09 01 _Calibration C 2009 06 24 C 2009 06 23 C 2009 06 15 E 2009 06 12 E 2009 06 10 Files adm2009 09 01_2022 001 adm2009 09 01_2022 002 adm2009 09 01_2022 003 l Open Cancel D o by s a a o g Analysis templates 5
194. sitions for grouping These sample positions must be ina column for example Figure 6 26 Only samples in a column can be selected In this example column 1 was selected and adjacent sample positions A1l B1 126 i 4 Click Group Group 5 Enter the sample information using the Experiment tab q admin File Edit View Mode Analysis Window Help Ce EEN PES Ke cS 0 a a OME Eerste Rack Chill 5 tube rack File adm2009 03 10 001 via E Project CD14 Analysis aos i odoodoodo O00000 000000 Figure 6 27 The above sample information applies to group 1 rack coordinates Al B1 only 6 Click on additional desired rack positions to perform further grouping Add sample information as required 000000 000000 000 000 Q 0O0O0000 Group 7 E Close the Rack dialog box 6 9 Working with data files in Custom mode Refer to the sections Opening files Saving files Importing files and Exporting files for immediate instructions on handling these file types If you are unfamiliar with the user interface or options associated with handling files read the following information Introduction to file handling 6 9 1 Introduction to file handling This section describes how data files can be opened saved
195. slot 3 Hold the column in one hand and gently unscrew the upper column connector counter clockwise Tilt the column downwards to empty any fluid Then 184 unscrew the bottom column connector Figure 9 2The top connector is removed from the old column 4 Insert the bottom end no white filter of the new MACSQuant Column into the bottom column connector and gently screw in the column by turning it clockwise until you feel resistance Point the column towards the top of the device and screw in the top column connector 7 Figure 9 3 The bottom column connector is attached to the new column before attachment of the top connector 5 Align the column so that the top column connector sits on the guide of the magnet cover Press the column into the slot until you feel the guides click Verify that the column is placed in the center of the magnet cover 6 Close the front door 9 1 4 Exchanging fuses If the instrument fails to start when switching it on or if operation suddenly stops and the screen is dark an exchange of the fuses might be required Follow steps 1 to 3 185 below to safely exchange the fuses 1 Turn the instrument OFF 2 Unplug the main power cord from the power outlet as well as from the instrument The fuse holder is located below the main power connector on the rear panel of the instrument 3 Pull out the fuse holder from the housing and replace with the appropriate fuses Replace t
196. ss and gateway Proxy IP address SMTP server E Software Mask Standard gateway DNS 4 Click Apply to implement changes Click OK close the window 6 11 Configuring the default user instrument and software options The Options menu is used to customize user software and instrument settings Custom users and administrators can customize software and instrument settings Only administrators have permission to modify user settings An explanation of the Options menu follows 6 11 1 Changing the default user options User groups Express Custom Administrator are managed using the Users menu File access permissions and the default location of files can be stipulated using this menu 138 To assign properties to a user group 1 Click Edit and Options Options ee Users ii Users Files Access Experiment E Instrument w Software Express Custom Administrator Group Password C Required Figure 6 37 The Users options window 2 Click the desired radio button Express Custom or Administrator 3 Check the Password box if a passport is required for this group 4 Click OK to save changes To assign properties to user file paths 1 Click Edit Options and Files Options Users Access Experiment E Instrument Public global E Software i Private luser Path Date File Zlnitials4 D ate Figure 6 38 Changing default
197. t gt 82 5 6 Working with data files in Express mode Refer to the sections Opening files and Saving files for immediate instructions or handling these file types If you are unfamiliar with the user interface or options associated with handling files read the following information Introduction to file handling 5 6 1 Introduction to file handling This section describes how data files can be opened saved and backed up in Express mode Data files may be stored to and therefore opened from a Public Private or External file location Public l Private External e Public files are located on the local hard drive of the MACSQuant Analyzer or personal computer and are accessible by all users e Private files are located on the local hard drive of the MACSQuant Analyzer or personal computer and are only accessible by the logged in user account e External files are located on an independent file storage device which is connected to the MACSQuant Analyzer or personal computer via the USB port i e a memory stick The default window for saving and opening data files is composed of the following tabs Figure 5 7 The default window for opening and saving various file types Note The availability of these tab options is dependant on the user profile Custom user Express user or administrator and whether data settings are being saved or opened Tab option Description The Workspaces tab allow
198. t Analyzer is equipped with light emitting diodes LEDs which illuminate each bottle to indicate the status of the instrument in Acquisition mode Green bottle light The instrument is ready to measure liquid levels are sufficient and the instrument is primed Note Please note that the lasers can take up to 30 minutes to warm up after performing the initial instrument priming Purple bottle light The instrument is measuring liquid levels are sufficient Blue light is indicative of normal instrument function during sample processing or that the instrument is busy Red bottle light Liquid level error general instrument error Red light indicates that the liquid levels are too low in a particular bottle or that the waste needs to be removed The bottle with the blinking red light will indicate which bottle needs to be tended to Additionally a message on the system status in the lower left corner will refill that a bottle change is needed A bottle can be replaced even during a measurement although replacing the waste is best when the instrument is not processing Yellow bottle light Sensor error Please ensure that the sensor is correctly attached to the bottle Note If no LED is illuminating the fluid containers then the instrument is in the Data Analysis mode and the lasers are not on Note Refer to section 6 1 for an overview of the MACSQuantify Software custom mode interface Before setting up an experiment the following q
199. t Analyzer USB port or computer USB port 132 Note If no external device is attached to the MACSQuant Analyzer or personal computer the following error will be displayed External i No external media found 2 Navigate to and highlight the file for export Workspaces Instrument settings Experiments Reagents Analysis templates Data files and Other files e g screenshots or windows bitmap lt bmp gt files can be exported g pO o oo i o 2 ES B __srv2008 12 10_2063 015 pos _st 2008 12 10_2063 013 ori E MC_CD4_T_Cell_Cocktail_h a E MC_CD3_Pan_T_Cell_Cocktai _st E MC_CD19_B_Cell_Cocktailh E MC_CD14_ Monocyte_Cocktail stv ag IL 1 E CD34cocktail hlog E CD34_CD133 cocktail log5 E CD34_CD133 cocktail hlog E CD34 Stemcell log5 E 2009 09 01 _Calibration EE 2009 06 24 C 2009 06 23 E 2009 06 15 4g 2009 06 12 vi rl 2 r 2 2 ee 2 RG lt fe 2 hy z S amp a 2 3 ra eagents ry Files v2008 12 10_2063 014 neg srv2008 12 10_2063 013 ori srv2008 12 10_2063 015 pos a g a 3 Click Export 4 The file will be exported 6 10 Data backup and restore in Custom mode It is recommended that data is regularly backed up to an external location Data can backed up to a network drive USB memory stick or DVD Administrators can configure data backup settings Please contact your administrator for more information or refer to section
200. t options The default Instrument options are the instrument name instrument features and instrument annotations Each are briefly discussed below To assign a default Instrument name 141 1 Click Edit Options and Instrument Options H Users Experiment a Features Annotations H Software Instrument Name Figure 6 42 Changing the default instrument name 2 Enter alphanumeric text into the Name field 3 Click Apply to implement changes Click OK close the window To assign a default Instrument features 1 Click Edit Options Instrument and Features Options E Users Experiment Instrument Annotations C Height Software Features Area C Width Use cale width Time C Use area for trigger C Use secondary trigger Figure 6 43 Changing the default instrument features 2 Click the checkbox to activate the corresponding feature 3 Enter desired alphanumeric text into the corresponding field 4 Click Apply to implement changes Click OK close the window To assign a default instrument Annotations Instrument annotations are used to provide additional description of the seven analysis channels that are available on the MACSQuant Analyzer Data analysis and interpretation is arguably easier when suitable annotations are used for example FL2 channel could be annotated as FITC as this fluorochrome is detected in this channel 142 1 Click Edit Op
201. t options Region functions Feature functions Region functions Unused In use Name Count Count l Count ml T Name The name axis name Count The actual total acquired events or count Count uL Number of acquired events per microliter Count mL Number of acquired events per milliliter Feature functions select and highlight a feature function and using the and buttons move the feature into the desired category Unused or In use Options Keyboard Timers Acquire Export Backup Regions Colors Windows Templates Views Statistic Feature functions Unused Mean StdDey Cy Min Max Median Modal Overlay Histogram Plot options Region functions Feature functions Mi 3 After making the necessary modifications click Apply to implement changes 4 Click OK close the window 6 12 Data analysis in Custom mode Acquired data are displayed and analyzed by an analysis window Depending on which New plot window template is applied by the user analysis windows may contain dot plots density plots histograms statistic and text tables Several analysis windows can be opened at one time These can be of several experiments or of a single experiment with a complex gating strategy Gating strategies can be created during sample acquisition Live and saved for future use or can be created post acquisition For background information c
202. th sufficient administration rights can save work to public or private folder Performing manual compensation Many experienced flow users prefer to set the compensation settings manually This can easily be accomplished by performing a standard measurement and adjusting the compensation values using the Classic or Matrix compensation menu 1 First perform automated compensation see section 3 7 1for more details 63 To access the compensation settings and change them manually 2 Open the Edit menu select Instrument settings and then click on the Compensation tab Instrument settings Channel Compensation Classic pier FL 2 3 FL 46 FL57 Figure 3 42 Two compensation modes can be selected from the Instrument Settings compensation dialog box Matrix left and Classic right 3 Use the drop down list to toggle between Matrix and Classic modes Matris Classic Note Converting data from Matrix to Classic formats is not lossless i e all changes made to the matrix may not be completely converted to the classic format The user should take care to ensure that manual compensation settings are always verified using acquired data i e labeled cells or compensation beads A Conversion not lossless 4 Reset the matrix by selecting Classic and then select Matrix 5 Change the matrix values to adjust cell populations as required See the following example for further clarification
203. th the exception of transient or stable expression of fluorescence proteins relatively bright fluorescence that occurs above background auto fluorescence requires cells to be stained with fluorescence dyes This is normally achieved through the use of antibodies that target a specific protein or other biochemical antigens expressed on or within cells These antibodies are either directly conjugated toa fluorochrome or can themselves be stained in a secondary step by a fluorochrome conjugated secondary antibody indirect staining Only cells expressing the particular target antigen will be fluorescence labeled After excitation by a laser light emitted from fluorescence dyes can be detected in defined wavelength ranges This differs from one fluorochrome to another The use of different light filters in the flow cytometer permits the simultaneous use of multiple fluorescence dyes and thus the detection of multiple cellular antigens These filters create fluorescence channels which is monitored by a photomultiplier tube PMT Each PMT which is located after a filter set will amplify the signal of the detected light Therefore the laser will excite a fluorescence marker on the cell which will be deflected by the cell and collected at a 90 angle This deflected light will pass through the appropriate filter and the resultant signal will be amplified and reported by the flow cytometer software i e the MACSQuantify Software It is strongly re
204. the Properties 6 i 7 option from the dropdown menu 1 Click the icon located adjacent to the histogram 2 Click Properties ee The properties window will appear Note Refer to Table 6 9 below for an overview of the various display Properties that may be modified for histograms using the MACSQuantify Software Tab Category Property Associated screenshot Description option View Data All All events are displayed on the histogram Percentile 50 v 1000 wa lt gt Percentile 50 25 5 2 or 1 percentile values of ee the total events can be displayed on the histogram This is useful when too many events have been acquired and the displayed plot histogram is saturated Fixed number l A fixed number of events can be displayed on u vla lt l gt the histogram Numbers can be entered directly into the field or by using the arrows Axes X axis xa s aote The x axis scale of a histogram can be E configured as follows As required automatically configured lin linear scale log2 5 logarithmic scales hlog biexponential scale Options Regions amn No regions None all regions All or only ere the selected region This can be shown on the histogram Normalization The histogram graphically summarizes the distribution of a univariate data set Data can be normalized by Area integral of total area under the curve or by Height Smoothing eom O
205. the orange solution basket prior to exchanging it Remove the cap and attach the bottle closure and sensor to the new bottle To empty the waste container remove the closure while the container is still in the holder close it with a cap Then remove the closed container Replace the bottle with an empty waste bottle and attach the bottle closure and sensor to the new waste bottle Note Handle the full liquid waste bottle with extreme caution and dispose of as recommended by your local authority Note It is recommended to add 100 mL of a MACS Bleach solution to the bottom of the waste container Note Check that all connections are securely fastened and that no tubing is tangled 6 3 5 Check the instrument status The instrument status can be monitored using the status bar and illuminated bottles 105 In order to start experiments the MACSQuant Analyzer should report the status Calibration Ok EA The following procedures must be completed before performing experiments on the MACSQuant Analyzer e Instrument hardware must be correctly installed and calibrated see section 3 5 e Instrument settings must be correctly calibrated and compensated see section 3 7 A more comprehensive explanation on monitoring the instrument status is given below Checking the instrument status using the status bar The instrument status is reported by the status bar using text and a corresponding color code Orange MACSQuant Analyzer
206. ther countries Ficoll and Ficoll Paque are trademarks of GE Healthcare companies BD and BD Falcon are trademarks of Becton Dickinson and Company All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only Unless otherwise specifically indicated Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use Software release 2 0 0945 R Copyright 2009 Miltenyi Biotec All rights reserved 1 Important information a aivasececcttacicadacstecdcesienddndesiaas gondeaatbadedonenacddeaisadientineteseasicet 9 eal SYMBOLS AND HAZARD LEVELS sciccsicaseateetercess cvecntadeantdanesersretercavesnataebanscee advaantensenaemeemeatenenncs 9 Vl id SetU Or saret NOCES sae v cach aa nd eunan a a A A awa seaees 9 kde SV ITI orrea a N a E A a a A IN E EAA eee 10 1 2 WARNINGS AND PRECAUTIONS lt c 2c ccc2ccarctscadeeteusoncedancenanaaeatee tee uent a E ari aii 10 3 GENERAL PRECAUTIONS cseuscacadts dunecdaccacctoecetsemeageanencoatacsectecsanes moumeaae ee eacne aul eand ones aa 11 1 3 1 Hazard of electric shock and spread of FC i succcccncccccecececenensnenenseesenesesenenensaeasases 11 V3 2 SOng magnetic Teld ireanii ie N E RE EN EA T ENE 12 1 3 3 Hazard OF crushing and SHCASING vusucucncceccececenenenenecensaeaceteretenenenseeaeesesesenenenenensases 12 13 4 Laser and LED FACQlaUlON urren nri EEE EEA RE ten nie EEE EEEN 13 LA SECURE I
207. therwise the calibration will fail Rack detection This step is required for the automatic detection of Chill 5 15 and 50 mL tube racks by the MACS MiniSampler 1 Ensure that the MACS MiniSampler is correctly attached to the instrument This includes fastening of the corresponding cable to the External CAN port located at the back of the instrument Note The single tube rack must be disconnected before performing this procedure Ensure that no objects are obstructing the MACS MiniSampler as this component will move during calibration 2 Select the Chill 5 rack from the Experiment tab and Rack dropdown menu Samples Experiment Tools Channels Experiment Rack Chill 5 tube rack File adm2009 09 07 001 va O Figure 3 32 The rack checkbox w must be activated in order to perform rack detection 3 Click Start calibration of the Rack detection 4 Place one Chill 5 tube rack onto the MACS MiniSampler The MACSQuant Analyzer will immediately move the Chill rack to and fro while checking the barcode reader for operability 55 5 A successful calibration is indicated by a green closed circle and the text report Calibration done File Edit View Mode Analysis Window Help admin x HES Ald E tout O Rack detection calibration equirements Single tube rack and Chill 5 rack the instruction in the specified cali ollow on the d ration on the right side Single tube rack and acquisitio
208. ting Strategies and open data files Previous window Close Close all Close a selected analysis window close IE aude all analysis windows Previous window Sequentially scroll through analysis Next window windows i e previous and next analysis window 100 Help Eg C ommand Description Open online help Help Open the preinstalled help file Info Info Information about current software version 6 2 User administration 6 2 1 Creating a new user In order to optimize the unique user management system of the MACSQuant Software it is recommended to create an individual user account for each user 1 Select Edit in the Windows pull down menu and User settings List of Users User Initials Group Rights Directory Public Private Password WER adm admin AXE C cap user adminIREAD IREAd yes Figure 6 2 The users dialog box 2 Click Add to create a new user Name New User Initials NU Administrator Public Private Instrument Settings Read v Read amp Write Experiments Read e v Read amp Write v Reagents Read v Read amp Write Analysis Read v Read amp Write d v Read amp Write Figure 6 3 Creating a new user account 3 Enter the Name Initials and associated access settings of the new user 101 Category Sub category Description User Name Enter the user identity in this field Initials Enter the respective initials Group Expr
209. tions Instrument and Annotations Options H Users Experiment Instrument Features FSC Fst H Software Annotations FL1 VioBlue FL2 FITC APC FL3 PE APC Cy FL4 PI PE Cy5 Figure 6 44 Changing the default instrument annotations 2 Enter desired alphanumeric text into the corresponding textbox 3 Click Apply to implement changes Click OK close the window 6 11 4 Changing the default software options The following default software options can be modified keyboard timers acquisition settings export and backup settings and the default display settings for regions plots histograms and tables Each software option is briefly discussed below To assign default settings for the Keyboard Note Feature only applicable to the MACSQuant Analyzer This function is not available to MACSQuantify Software installations on personal computers Figure 6 45 Changing the default keyboard settings 2 Use the dropdown list to select a keyboard format The opacity can be modified as required 3 Click checkbox Show at start to automatically activate the touchscreen keyboard at MACSQuant Analyzer startup 4 Click Apply to implement changes Click OK close the window 143 To assign default settings for Timers Note Feature only applicable to the MACSQuant Analyzer This function is not available to MACSQuantify Software installations on personal computers Options E
210. to define an area of interest Ellipse rectangular and polygonal regions can be drawn Draw a quadrant in a dot plot Draw an interval ina histogram Open a new analysis window Close analysis window Scroll through open analysis windows in a reverse and forward direction C Logout admin Description Activate the Analysis template tool Scroll through samples listed in the samples window Backup or transport data Activate the reagent barcode scanner Open the rack dialog box Open the instrument settings dialog box Activate touch screen keyboard Open help file and manual Switch to Express mode Logout user from session Main instrument control Click to switch between Acquisition mode or Data analysis mode The instrument may also be switched off using this button Name of user in the top right corner in this example the administrator admin is logged in 98 6 1 2 Quick guide to the MACSQuantify Software menus File Edit View Mode Analysis Window Help Command Description New workspace Ctrl M New workspace Create a new workspace The user will Open Chr be prompted to save any changes to the workspace before this action is Save Chrl 5 performed Import Experiments Analysis templates and or Data files depending on the user access Export rights set by the administrator i pat pen Min Open Open Workspaces Instrument Settings
211. to the bottle closures are tightly sealed If the problem persists contact the Technical Service 10 1 6 Excessive debris is present in acquisition Check all fluidics to ensure that all tubes are connected properly Ensure that the pre filter is bled of air 189 Additionally you can rinse or clean the system Push the rinse button teardrop Before this twice If this does not work try to clean the system by 10 1 7 Touchscreen remains dark 1 Check if the power cord is plugged in correctly and the power supply is switched on 2 If the device is powered up and the touchscreen remains dark switch off the device wait 5 seconds and switch on again If the MACSQuant Analyzer still does not initialize go to step 2 3 Replace the fuses Spare fuses are included in the MACSQuant Analyzer Starting Kit this is not true If the problem persists contact the Technical Service 190 11 Hardware monitor Using the Hardware Monitor the current hardware status of the MACSQuant Analyzer can be assessed at any time The hardware pages can provide additional information on the status of the instrument in the case of error messages appearing on the screen Never run the instrument if any errors are indicated within the hardware monitor 11 1 Hardware Monitor window The Hardware Monitor window can be accessed under the View menu The window contains five tabs each displaying the hardware components involved in the respective proc
212. to the sensor cable port 7 Note the color coding and connect each sensor cable to the respective bottle closure Sensor cables Air filters 8 Connect the hydrophobic air filters 0 2 um to the appropriate connectors on the bottle closures Note The correct positioning of each solution container recognizable by the color code and the symbols is crucial for successful analyses using the MACSQuant Analyzer To keep buffer sterile each bottle closure should be equipped with a hydrophobic air filter Avoid any contact of hydrophobic air filters with fluids as this may cause clogging of the filter When working with biohazardous samples it is recommended to fill the waste container with 100 mL of disinfectant e g MACS Bleach solution order number 130 093 663 before use For proper disposal please follow local regulations and carefully read the chapter Important information 37 3 2 2 Connecting the power cord 1 Note the position of the power socket on the rear panel of the MACSQuant Analyzer Ensure that the main power switch is in position O before connecting the power cord 3 2 3 Installation of the MACS MiniSampler and tube racks and reagent rack The MACSQuant Analyzer is optionally delivered with the MACS MiniSampler three different tube racks and a reagent rack MACS Reagent Rack 4 Once installed the MiniSampler is automatically recognized by the MACSQuant Analyzer Each tube rack
213. ts Maximum Option on Corresponding MACSQuantify number of MACSQuantify Rack Rack Graphic samples drop down list 1 5 mL Single tube rack x tube Not applicable x50mL 3 50 mL 3x15mL tubes 3x5 mL 96 well O lw eh EY Ele a Th ae microtiter plate Table 6 5 Overview of the various rack types that may be used with the MACSQuant Analyzer An appropriate rack should be used depending on the sample number and volume 6 7 1 Selecting a sample rack Note This information is also relevant to section 6 3 6 Define an experiment in Custom mode of this manual To select a sample rack 1 2 3 Click on the Experiment tab Select the required rack type from the Rack dropdown list Rack Single tube rack v File Single tube rack Chill 5 tube rack Experiment Chill 15 tube rack Sample ID Chill 50 tube rack ap es re 96 well The corresponding rack template will popup in an independent window and will also appear in the lower section of the Experiment tab window mq admin File Edit View Mode Analysis Window Help admin aala adoa ae 5 ali Ome Sl es e e 0 Samples Experiment Tools Channels Experiment Rack Chill 5 tube rack vio Fie ma w Poet SSS d smeo OOo E0 Description E Flow rate Se 1 2 3 4 5 6 Pickup and measure Liquid sensor C Mix sample O O O C C O Mode Standard v Uptake vohie o Sewing OO E ESZOTS T
214. tube z axis i e almost touches the tube bottom To check the needle position relative to the bottom of the tube gently wiggle the tube to ensure that there is a small amount of movement If this is not the case carefully adjust the needle arm accordingly 7 When satisfied with the needle position click Save calibration 8 A successfully completed procedure is indicated by a green closed circle 9 Click Test to confirm that the correct coordinates are saved The MACSQuant Analyzer will automatically test the remaining sample positions File Edit View Mode Analysis Window Help x x e les Experiment Tools Channels ampl T Calibrate uptake unit brat Uptake unit calibration bi to begin the calibration be carel e hardware moves b reful the hardware movi e specified position and press Save calibration position you want to test Needle arm calibration relative to washing station Zoftset 0 Start calibration Home pos 0 00 mm Measure pos 29 10 44 50 mm Single tube rack Zoffset 0 Start calibration Save calibration Test Offset 64 90 51 10 3 30 mm Chil 5 tube rack Zoffset 0 gt Start calibration Save calibration Test Offset 13 60 44 70 0 10 mm 96 well Offset 13 90 45 30 4 00 mm Reagent Rack 4 z 0 Start cali Offset 11 60 45 40 4 00 mm j Tine Voorn Fate Count 10 56 Ol Data analysis wode i 10 58
215. ty versus cell count B In Figure 2 4 B the signal intensities of non fluorescence cells and fluorescence labeled cells are squeezed together To separate the signals a log5 scale was used Figure 2 4 C revealing two peaks the left peak is attributable to background fluorescence whereas the right peak is due to cells labeled with PE As the MACSQuant Analyzer acquires data in a digital format some fluorescence intensities may be assigned a value less than zero Data values less than zero may not be displayed properly using a conventional logarithmic scale although 26 however all calculated statistics will be correct This is a general feature of more advanced digital flow cytometers To overcome this a hyperlog Hlog or biexponential scale may be used D In a Hlog scale the upper values of the scale are logarithmic whereas the lower values are linear 2009 01 30_2062 008 _ 1 30_2062 0084 P2 baa me Ai feo i 750 500 400 FOO a00 oe 200 250 a a WY hate Saar ee Se Sag Ts eA 100 o oO T 0 Tj 2 0 250 500 750 1000 0 50 500 50 1000 FSC A D PE A Linear Linear 1 30 _2062 0085 4P2 1 30 _2062 0085 4P2 100 Cy i a0 i 70 75 BU a BO 40 30 25 20 E Z 10 aj alo 5 1 0e 4 0e 11 0e0 1 0e1 1 0e2 1 0e3 4 0 1 1 0e1 1 0e2 1 0e3 a PE A C PE A Logs Hlog Figure 2 4 Comparing log and linear scales when displaying fluorescence and non fluorescence events A Dot plot showing forwa
216. uestions should be addressed 1 How many samples will be analyzed and what is the sample volume Single samples are processed using the Single Tube Sample Rack Multiple samples up to 96 are processed by using the MACS MiniSampler in combination one of the following Cooling Tube Racks 107 Rack type Slots Chill 5 24 x 5mL Chill 15 15x15 mL 5x5mL 6 Chill 50 x 50 mL 3x15 mL 3x5 mL Chill 96 rack 96 rack 96 well microtiter plate Choose the appropriate rack and configure it menu accordingly Samples Experiment Tools Channels Experiment Pak N O File adm2009 09 08 001 vial Project O Sample ID O00000 F Description Note Refer to section 6 8 for information concerning use of the MACS MiniSampler and configuration of sample racks Multiple samples up to 96 are processed by using the MACS MiniSampler in combination one of the following Cooling Tube Racks 2 Will autolabeling be performed or is sample analysis only required Yes autolabeling is required Up to four MACS Reagents can be placed on the MACS Reagent Rack 4 for 1 Magnetic autolabeling of rare cell populations for subsequent pre enrichment and analysis 2 Fluorochrome autolabeling of specific cell populations No autolabeling is not required If samples are manually labeled no MACS Reagent Rack is required For manual labeling follow the labeling instructions in the corresponding datasheet 3 Are rare cel
217. uipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instructions may cause harmful interference to radio communications However there is no guarantee that interference will not occur in a particular installation If this equipment does cause harmful interference to radio or television reception which can be determined by turning the equipment off and on the user is encouraged to try to correct the interference by one or more of the following measures Reorient or relocate the receiving antenna Increase the separation between the equipment and receiver Connect the equipment into an outlet on a circuit different from that to which the receiver is connected Consult the dealer or an experienced radio TV technician for help 2 Introduction 2 1 Purpose The MACSQuant Analyzer is a benchtop flow cytometer that has been specifically designed for the rapid simple and automated fluorescence analysis of single cell suspensions The MACSQuant Analyzer also facilitates the absolute quantitation of cell populations and has a processing rate of up to 10 000 events per second The instrument was designed for use with MACS Cell Analysis and MACS Separation Reagents in research applications though common fluorochrome conjugated antibodies from other suppliers can also be used The relatively small footprint of the MACSQuant Analyzer 60x35x40 cm in comparison to other commerc
218. users to save user an entire workspace which is composed of instrument settings experiment and reagent definitions and an analysis template with accompanying data Instrument settings are compensation and calibration parameters for the MACSQuant Analyser These parameters are important for data analysis and are vital to maintain standardized results over time and from instrument to instrument The MACSQuantify Software can open and save instrument settings These settings can be applied to acquired data and thus this useful feature allows users to perform recompensation after data acquisition 83 Be The Instrument settings may be saved but not opened in Express mode Experiment definitions can be saved for future use Reagent type and corresponding Reagent Rack 4 positions sample rack type and corresponding Chill Rack sample positions the analysis mode and sample processing definitions e g labeling strategy comprise experiment definitions Reagent type and position on the reagent rack can be saved using the Reagents tab The Reagents tab is not available in Express more Analysis templates are predefined analysis layouts for data acquired by the MACSQuant Analyzer The templates are created by defining a gating strategy with associated plots histograms tables and statistics Administrators and Custom users can customize and save templates for reuse Express users cannot create or modify Analysis templates Data files ca
219. ut of data analyzed by the MACSQuantify Software in Custom mode 6 6 Reagent management Reagents are positioned on the reagent rack There are four positions for reagent vials on the MACS Reagent Rack 4 Reagents can be entered using the 2D code reader or manually using the Reagents window 114 H Reagents q Pas Category Reagent Time Titer Order RI Human MB v CD14 Flucrochromes x 10 vfa 1 11 vjal 3 val li Or2 H CD14 F 10 va 4 11 vf 3 via G OR3 H E 10 vja 4 11 vf 4 3 vlaj fi O R4 H 10 viol 1 11 viel 3 vial i g s pecial f 0 viol 10 1 vis 5 via i o s2 pecial o xja 101 xla 5 vial i ss Figure 6 14 The reagent rack Reagent positions 1 2 3 and 4 are clearly marked on the MACS Reagent Rack and correspond to positions R1 R2 R3 and R4 of the MACSQuant Analyzer reagent management window 6 6 1 An overview of the MACSQuant Analyzer reagent management window The MACSQuant Analyzer reagent management window allows users to select reagents from a dropdown list and assign reagents to a position on the reagent rack The available options are described below E Reagents m o Pos Category Reagent Time Titer Order AT HumanMB v CD14 Fluorochomes v 10 wla 1 11 vial 3 vial li R2 Human MB CD14 Fluorochromes 10 vja 1 11 lal 3 vja G C R3 Human MB CD14 Fluorochromes 10 xja 1 11 yal 3 vja G C R4 Human MB CD14 Fluorochromes 10 vial 11
220. utomatically written to the network drive 5 11 Logging out from Express mode Logout 1 Click the Logout icon 95 2 If prompted to continue click OK The software will return to the login menu MACS Quantify Figure 5 23 Login menu 5 12 How to close the MACSQuantify Software Note This information is only applicable to personal computer users of the MACSQuantify Software Please refer to the chapter How to shutdown the MACSQuant Analyzer for instructions on how the instrument may be cleaned and shutdown O 1 Click the Shutdown close software icon 2 Click Exit to continue closing the software Click Abort to continue working with MACSQuantify Software Exit MACSQuantify 96 6 1 6 Custom mode The Custom mode is designed for advanced flow cytometry users Administrators and Customer users can use the Custom mode interface to create customized experiments ranging from sample autolabeling and uptake through data acquisition gating and data analysis to the generation of print ready results Custom users and administrators have advanced access to MACSQuant Analyzer instrument and software settings Administrators have additional permissions concerning setting user permissions and the management of Express and Custom mode users Both administrator and Custom user features are discussed throughout this chapter Custom mode quick reference guide The quick referenc
221. v Uptake volume 50 ul al Sample volume 200 pl C Annotations Autolabel Settings Custom Express Type Setup v Mode A Figure 3 40 Setting up compensation from Custom mode using the Express radio button 62 4 Click on the Start Measurement button 2 This will start the automated compensation process A dialog box will appear to describe that compensation will only be performed on the default settings An analysis template is automatically generated in Live mode for each of the previously selected channels Initially the MACSQuant Analyzer acquires 2 500 events before determining the compensation values for the channels the instrument will continue to acquire events until all of the channels have been compensated All newly acquired settings for the compensation are automatically saved in the compensation matrix 5 The newly saved instrument settings will automatically be saved into the current default settings The calibrated and compensated settings are now saved as the default settings for the day s date 6 Save the instrument settings either to a Public or Private folder Only save the instrument settings if the compensation was correctly performed Select File and Save from the menu Ctrl S Highlight Instrument settings on the left navigation bar enter the filename in the Setting field and Save the settings The file is automatically saved Figure 3 41 Saving instrument settings Users wi
222. verlap in emission spectra observed between different fluorochromes This fluorescence spectral overlap or spillover will result in the detection of individual fluorochromes in more than one fluorescence channel This overlap should be determined and corrected This is especially important when multicolor analyses are to be performed without proper compensation results may be misinterpreted For example FITC fluorescence will be detected in both the FL 2 FITC and FL 3 PE channels though FL 2 is the designated channel for FITC detection Therefore all FITC fluorescence signal detected in FL 3 is regarded as excess or spillover this excess is calculated as a percentile the value of which is mathematically subtracted compensated from the original signal This is best displayed by Figure 3 38 where cells labeled with a CD8 FITC antibody are measured in an FL 2 vs FL 3 dot plot The left panel shows an uncompensated detection of the cells where CD8 cells are detected in both the FITC channel FL2 and the PE channel FL3 and hence appear to be double positive for FITC and PE fluorescence even though they have only been stained with a FITC conjugated antibody After compensation right panel the percentage overlap between the two channels has been removed ensuring that FITC signal from CD8 labeled cells are restricted to the x axis FITC 2008 03 14 0044P1 Lies ee FL 3 A 1 0e 2 10e1 1 0e
223. vial 3 vial i 181 Special PreBuffer o vja 10 1 vja 5 vial i 182 Special PreButfer o vja Doya 5 fa Figure 6 15 The MACSQuant Analyzer reagent management window e Pos Use this checkbox to assign reagents to rack positions R1 R2 R3 R4 S1 or S2 o R1 R4 positions are located on the MACS Reagent Rack 4 S1 and S2 positions are denote as Special positions where the Running Buffer is taken directly from the buffer bottle e Category Reagents are categorized according to species conjugated fluorochrome and purpose The current categories follow o Calibration MACSQuant Calibration beads for calibration of the instrument settings o Species and conjugated fluorochrome e g Human APC Mouse PE o Isotype control isotype control antibodies are raised against non mammalian epitopes and can be therefore used as a negative control for non specific binding o MACS Comp Reag MACS Compensation Reagents These reagents are used to correct the inherent spectral overlap between excitation and emission wavelengths of fluorochromes o Universal for generic labeling strategies using Tags such as biotin of His 115 e Reagents A dropdown list of available reagents is displayed in accordance with the selected category for example the following reagents are available for the category Human Cocktails MC CD14 Monocyte Cocktail human v MC CD14 Monocyte Cocktail human MC CD19 B Cell Cocktai
224. ware dialog box 4 Place the reagent onto the corresponding position on the reagent rack Note When scanning MACSQuant Calibration Beads the instrument will prompt to initiate the calibration procedure Scan result i 5 Calibration Particles detected LOT 5080414001 Would you like to proceed with calibration process Note When scanning MACS Reagents the MACSQuantify Software will prompt the user to place the vial s on the MACS Reagent Rack Note If the code reader fails to recognize the 2D code enter the information directly into the MACSQuantify Software reagent management window see section 6 6 for more details 117 6 7 Multisample processing Multisample processing is accomplished by use of the MACS MiniSampler in combination with the MACS Reagent Rack 4 and MACS Cooling Tube Racks sample tube racks Five different kinds of sample tube racks are available Single tube rack Chill 5 Chill 15 Chill 50 and Chill 96 allowing for processing of up to 96 samples ina single batch see Table 6 5 for details The MiniSampler can be configured to perform measurements from any sample position Depending on the user s instructions samples can be labeled with fluorochromes and or measured Rack type Chill 5 Chill 15 Chill 50 Chill 96 rack 96 rack 24x5mL 6 5 mL Eas tubes peoeeee 15x15 mL 5 15 mL Fon aes Pavavrereava Sx5 mL tubes ey ay Q a C SOOO O0O SOOQO00O 6 Slo
225. will automatically log into the Express mode If the user has been registered as a Custom mode user the MACSQuantify Software will automatically log into the Custom mode Note Please contact your administrator if there is no suitable user name and or the password is incorrect User s Password Figure 5 2 Logging in the user Express User to the MACSQuantify Software in Express mode 5 3 Switching to Express mode from Custom mode 1 In Custom mode click Express mode button in the top right hand of the navigation bar Qm HE Ss Figure 5 3 Switching to Express mode from Custom mode 2 The MACSQuantify Software window will change to the Express mode Note If windows are active in the Custom mode e g analysis window the user will be prompted to confirm this action Click Yes to continue and No to cancel the switch 76 Note Any active work will NOT be transferred to the Express mode All data or settings must be saved before switching to Express mode 5 4 Using the touchscreen keyboard on the MACSQuant Analyzer The touchscreen keyboard can be used to enter information into the Sample ID and Description fields Users may find it easier to use a conventional keyboard and mouse which may be connected to the back of the analyzer as described in section 12 1 To activate the touchscreen keyboard perform the following 2 The Keyboard popup window will appear
226. will be automatically logged into the Express mode If the user is set as a Custom mode user the user will be automatically logged into the Custom mode window This same applies to administrators 6 3 Getting started in the custom mode This section is intended to provide the user a quick overview of actions required for a quick start of the MACSQuant Analyzer in the custom mode Some familiarity with the MACSQuantify Software is assumed 6 3 1 Turn on the instrument Switch on the MACSQuant Analyzer by pressing the touchscreen monitor while it is in standby mode The MACSQuant Analyzer will automatically check and initialize the system following which the log in screen will be displayed 6 3 2 Login as administrator or custom user 1 Switch on the analyzer 2 At startup a dialog box will appear prompting you to select your username and password The administrator admin account should be used for the first log in attempt MACS Quantify Figure 6 5 Logging in for the first time as an administrator A dialog box prompts the user to confirm the password Note Custom users must only select their login name from the User dropdown list No further action is required 3 Select user account admin and enter a new password Select log in 103 4 After a successful log in the software will automatically prepare the instrument for analysis The status of this startup procedure is indicated by the System setup di
227. with running buffer for use during cell analysis a washing solution for rinsing the instrument and a storage solution for overnight or long term storage A fourth bottle is used to collect all waste produced during operation of the instrument Fluid levels within each container are constantly monitored by sensors contained within the bottle closures and a warning message appears on screen when action is required by the user Solenoid valves Valves Flow cell Pressure sensors MACSQuant Syringe pumps Column MACS Enrichment Unit Figure 12 3 Front of MACSQuant Analyzer with access cover removed 197 Sockets for keyboard and mouse cece oo Guiding for sensor cables and tubing Fan Sheath particle Socket for sensor cables filter Socket for code reader Socket for MACS MiniSampler Socket for main plug Figure 12 4 Rear view of the MACSQuant Analyzer Air filter The fluid particle filter provides a physical barrier to prevent debris larger than 70 um in size from entering the fluidics system from the system buffer cleaning solution containers The fluid particle filter can easily be exchanged when blocked or at regular intervals to prevent blockages Plugs connections and guides Sockets for the main power supply the fluid sensor cables and the MACS MiniSampler are located at the rear of the instrument Figure 12 4 Extra sockets have been provided for the case of further instrument development The main pow
228. with skin and eyes Also protect mouth and nose as aerosols might leak from the system e g Washing Station Defective or inadequate safety equipment might endanger the operator The MACSQuant Analyzer shall be operated in a safety hood if hazardous or unknown materials are processed If hazardous material has been used or spilled care must been taken to thoroughly decontaminate the system For details see section 9 1 9 Always inspect the fluidics system complete tubing set reservoirs bottles and their closures valves columns diluters peristaltic pumps and needle before switching on the device If leakage has been detected replace all damaged parts before switching on the device If damaged parts cannot be replaced unplug and do not use the device Failure of parts containing biohazardous material or liquids that have been in contact with such material could cause a hazard Columns tubes and any other consumables that were in contact with biohazardous samples shall be autoclaved prior to disposal Liquid waste shall be autoclaved or decontaminated using a disinfectant that is appropriate for the specific pathogen e g 10 bleach isopropyl alcohol or 70 ethanol Miltenyi Biotec recommend use of MACS Bleach Waste disposal must be in accordance with any local regulations with 21 CFR 1040 10 and a ace fer deviations pursusni to fe Motes 50 dated July 26 2001 ectured Merch S007 Bictes GmbH F remal rith E ber ir 68 14
229. y performed maintenance or repairs accident acts of God limitations of technology electrical current fluctuations modification of or to any part of the Product use of accessories spare parts and or consumables other than those recommended by Miltenyi Biotec or normal wear and tear Miltenyi Biotec s product warranty does not cover products sold AS IS or WITH ALL FAULTS or which had its serial number defaced altered or removed or any consumables or parts identified as being supplied by a third party those third party accessories or parts may be covered by a separate warranty from their manufacturer Miltenyi Biotec must be informed immediately if a claim is made under such warranty If a material or manufacturing defect occurs within the warranty period Miltenyi Biotec will take the appropriate steps to restore the full usability of your Product Limitation on damages Miltenyi Biotec shall not be liable for any incidental or consequential damages for breach of any express or implied warranty or condition on this Product Some states or jurisdictions do not allow the exclusion or limitation of incidental or consequential damages so the above limitations or exclusions may not apply to you This warranty statement gives you specific legal rights and you may have other rights which may vary from county to country or jurisdiction to jurisdiction 207 15 Air filter Air filter connector APC MACSQuant Column MACSQuant
230. yed for well resolved homogeneous populations or for comparing intensities of multiple samples for a single parameter overlays 1000 100 foo fo 500 SSC A a 250 2 0 250 500 750 1000 1 0e2 1 0e1 10e0 1 0e1 1 0e2 1 0e3 FSC A CD3 PE A Figure 2 2 Left FSC SSC dot plot of PBMCs that were stained with CD3 antibodies conjugated to PE CD3 cells are depicted green Right Corresponding histogram of the CD3 cell population fluorescence intensity x axis is plotted against relative cell number y axis Density plot Density plots are a useful tool With traditional dot plots it may be difficult to quickly determine the intensity or frequency of acquired events on a black and white graphic A density plot is plotted in grayscale or in color each color shade provides information about the intensity of acquired events In essence the density plot is designed to represent a three dimensional plot where the number of cell events are depicted in a third dimension either by shades of grey or by using different colors 25 66C A 0 a 500 fou 1000 FSC A Lymphocytes Figure 2 3 Representative density plot of peripheral blood mononuclear cells The red color represents the highest density of cells followed by yellow then green and finally royal blue which represents the cells occurring at the lowest frequency Statistics Before performing statistical analysis of cell populations it is important to note that th
231. ytometry 122 This can be easily and quickly achieved using the MACSQuant Analyzer in combination with the MACS Control MC CD14 Monocyte Cocktail human Three samples were placed on rack positions Al A2 and A3 of a Chill 5 tube rack Cells in sample tube positions Al and A2 were analyzed using the MACS Control MC CD14 Monocyte Cocktail in the Express mode Cells in sample tube position A3 were analyzed in the Custom mode using an analysis template The default instrument setting was used i e the most recent instrument setting which is defined as Default Banks Note See sections 3 6 and 6 12 for more information on Instrument settings and analysis templates respectively 6 8 2 Rack configuration and sample definition 1 Choose Chill 5 tube rack from the Experiment tab mq admin File Edit Yiew Mode Analysis Window Help aala ods elo bd It u INTO MECESDHEGE Rack Chill 5 tube rack File adm2009 09 08 001 v a Project Sample ID Description Flow rate Low Medi High Pickup and measure Liquid sensor C Mix sample Mode Standard Uptake volume 100 pl LJ Sample volume 200 ul C Annotations Autolabel Settings FLI VioBlue FL5 PE Cy7 FL2 FITC FL6 APC FL3 PE FL APC Cy FL4 P

Hotlink to MACSQuant User Manual

Contents

Download Pdf Manuals

Related Search

Related Contents