FlowSight user manual
Contents
1. This section covers safety information for operating the Amnis FlowSight flow cytometer Anyone who operates the FlowSight should be familiar with this safety information Keep this information readily available for all users The FlowSight imaging flow cytometer is manufactured by Amnis Corporation and has a rated voltage of 100 240 VAC arated frequency of 50 60 Hz and a rated current of 1 5 A The years of construction were 2011 2013 and the product contains CE Marking Environmental conditions This instrument was designed for indoor use at an altitude of less than 2000 m at a temperature from 5 through 25 C and at a maximum relative humidity of 80 non condensing During instrument operation the ambient temperature should be maintained within 2 C The mains power Supply may not fluctuate more than 10 and must meet transient over voltage category Il The instrument is evaluated to Pollution Degree 2 Noise level The noise level of the FlowSight is less than 70 dB A Weight 56 kg Ventilation Provide at least 3 inches of clearance behind the instrument to maintain proper ventilation Disconnection To disconnect the instrument from the power supply remove the plug from the socket outlet which must be located in the vicinity of the machine and in view of the operator Do not position the instrument so that disconnecting the power cord is difficult To immediately turn the machine off should the need arise remo2. Center for Devices and Radiological Health 21 CFR Chapitre 1 subchapitre J Aucune radiations laser sont accessible a l utilsateur pendant le fonctionnement normal Quand le capot est ouvert les enclenchements eteindents les lasers FlowSight peut avoir les lasers suivants Table 3 ongueur d opnde La Puissance Maximale 00 413 nm 150 mW 83 493 nm 58 562 nm 35 647 nm 150 mW 75 795 nm Les etiquettes d avertissement suivantes sont placees dans l interior A DANGER Appareil au laser Classe IIIB radiations laser quand ouverte et les enclenchements sont defaites Ne regarder pas la source du faisceau Les etiquettes d avertissement suivantes sont placees dans L Interieur de panneaux lateraux pr s de mecanismes de liberation Avertissement L utilisation des commandes ou les rendement des procedures autres que celle precisees aux presentes peuvent provoquer une radioexposition dangereuse Chapter 1 General Information and Safety Biological safety Biohazards The FlowSight is rated at BSL1 Do not load or flush samples containing infectious agents without first exposing the sample to inactivating conditions It is recommended that samples be fixed in 2 paraformaldehyde for at least 10 minutes before running the samples on the FlowSight The use containment and disposal of biologically hazardous materials are required to be in accordance with Personnel Protective Equipment Directive 93 95 E and are the res
3. Definition def that assigns instrument settings to wells names to the sample output files and parameters to include in the plate report see procedure below Add 75 vl samples to the wells of a u bottom 96 well plate and cover with Sigma Aldrich X Pierce Film XP 100 Cat 2722502 and load the plate into the auto sampler Run the plate see procedure below Define the plate The steps required to create a Well Plate Definition def and run a plate are outlined below 1 2 3 Choose Define Plate from the Autosampler menu to open the Well Plate Def inition window UM ee tee AA ee E RE SE RE NET 4408 0 0 SS DING 0 0 0 0 9 Li seal alezezz HI LT ee 1440400 0 1 0 BO 0 0 0 0 eee 4 BABAE AA ANN 199 0 0 0 0 0 0 0 0 9 US ST FSR RE eas SZ Browse for a previously saved definition to edit by clicking on the folder icon or continue to create a new definition Name the plate definition 36 Autosampler option 4 Ataminimum each well requires an Output File Path Max Acquisition Time and Template File in order to be considered defined Optional parameters can be added to the definition in the next step e Toinclude a parameter in the file name click in the box below the column heading make sure it says yes e Columns can be re ordered by click drag 5 Click Add Remove Well Parameters to choose the parameters you want to report for the wells There are sev
4. Input Current 216 A per phase IEC 61000 3 2 2008 edition EMC Part 3 3 Limitations of voltage changes voltage fluctuations and flicker m public low voltage supply systems for equipment with rated current lt 16 A per phase and notsubject to conditional connections IEC 61000 3 5 1965 edition Electromagnetic Compatibility Part 4 Testmg and Measurement Techniques Section 2 Electrostatic Discharge Immunity Test EN 61000 4 2 2008 edition Electromagnetic Compatibility Part 4 Testmg and Measurement Techniques Section 3 Radiated Radio Frequency Electromagnetic Field Immunity Test EN 61000 4 3 2008 edition Electromagnetic Compatibility Part 4 Testmg and Mezsurement Techniques Section 4 Electrical Fast Transtent Burst Immunity Test EN 61000 4 4 2004 edition Electromagnetic Compatibility Part 4 Testmg and Mezsurement Techniques Section 3 Surge Immunity Test EN 61000 4 5 2005 edition Electromagnetic Compatibility Part 4 Testmg and Measurement Techniques Section 6 Immunity to Conducted Disturbances Induced by Radto Frequency Fields EN 61000 4 2008 edition Electromagnetic Compatibility Part 4 Testmg and Measurement Techniques Section 5 Power Frequency Magnetic Field Immunity Test EN 61000 4 5 2001 edition Electromagnetic Compatibility Part 4 Testmg and Measurement Techniques Section 11 Voltage Dips Short Interruptions and Voltage Wariations Immunity Test EN 61000 4 11 2004 edition Safety Requirements f
5. bright as possible IgG orescent capture beads or a cell line stained with a single fluorochrome may be used for comp controls Compensation controls must be a sam ple with a single fluorochrome label in a single tube Each fluorochrome must be run separately Cells are stained with more than one fluorochrome Cell concentration is too low when Compensation controls must be running using the com at 25 cells per second at a minimum pensation wizard Software Symptom Possible Causes Recomended Solutions INSERE appears Camera aNG TON Se to freeze ning 50 Chapter 4 Troubleshooting Symptom Possible Causes Recomended Solutions If the camera is Click Stop then Run Setup o D Q Q lt c 5 5 Imaging is paused Click Resume In the cell view area select the all pop ulation stop the operation described If a crash occurs during the day a complete shutdown is rec ommended at the end of the day prior to running the sterilize script On the keyboard press Ctrl Alt delete INSPIRE fails to Splash screenis lopen the task manager select INSPIRE i Open the Windows Task Manager by simultaneously pressing lt Ctrl Alt Del gt Click the Applications tab If the INSPIRE for FlowSight status is Not Responding select the INSPIRE task and click End Now Restart the INSPIRE application by double clicking the FlowSight icon on the desktop If the program restarts
6. concentrations in Current Protocols in Cytometry should work Instrument is expe ae Allow the instrument to warm up by riencing large tem running for 15 minutes Direct a fan toward the back of the instrument to dissipate excess heat or move the system to a temperature con trolled environment Run the purge bubbles script from the instrument drop down menu See solu tions for unstable fluidics Large variation in Run the purge bubbles script from the MG Large flow speed brightfield intensity instrument drop down menu See solu variation due to air a napasama levels tions for unstable fluidics 57 Chapter 4 Light source deliv Power down and power up the instru ering variable out ment if this does not fix the problem put call Amnis service Brightfield intensity Intensity set before Allow the system to stabilize after load level sets incor desired flow speed ing a sample and then click Set Inten rectly has been achieved sity Power down and power up the instru Movable optics are ue an ment if this does not fix the problem out of position call Amnis service _ 58 Index Index Aggregation 10 Analysis Area 15 tools 15 Area 31 Aspect Ratio 31 Autosampler 36 Bkgd Mean 31 Bkgd StdDev 31 Calibration 21 Calibration Beads 19 Camera Line Number 31 Camera Timer 31 Cell concentration 9 Cleanser 19 Compensation samples 10 wizard 26 data f
7. followed by sample runs and data acquisition and finally a shutdown procedure which sterilizes the system and prepares for the instrument for use the following day Optimizing instrument setup for sample runs is also described in this chapter User Interface Reagents Daily Calibration and Testing Instrument Setup Data Acquisition Daily Shutdown Autosampler Quantitative Imaging 13 Chapter 3 FlowSight User Interface The user interface is divided into 3 areas the image gallery where channel images are displayed a work area where graphs of features are displayed and the controls section where the instrument is controlled Status information is displayed along the bottom of the window Sont x INSPIRE for Flowsigh ngin e File Instrument Analysis Compensation Layout Advanced Help 104 S WE kRtHttbd Beas EA A Sample mi M3 Ma Ch5 m6 h7 ms chs ji cno mit miz All R e3 1e4 1e5 Intensity MC Ch02 Work Area mm uEle NEE Instrument Pamatay Controls ANE amni part of EMD Millipore Startup Shutdown Run Sample completed at 10 38 AM on 8 3 2011 Shesh am Flush m 488 O 7350 EF Compensation Flow ASSIST Count 2 094 043 The Image Gallery Images are displayed in the image gallery during setup and acquisition Image Gallery Tools All 453 obj s Il aa PAA TY Choi Chiz Ch03 Chos chos chos Cho7 Choe An Select the population to vie
8. scien croc MN Ur punan upod 0 Technical Assistance 2 0 Table of Contents i Chapter 1 General Information and Safety 1 Declaration of Conformity 2 22 0 aaa 3 Explanation of symbols 22000 eee cece cece ec eececcecceeceecteceeceeceeeteetecees 4 El ctical safey AA nn OR a a ee Wk 5 S curit Electronique 2 5 Laser Safety idee 5 S curit Laser lili didier 6 ka tor Kea CE ee E nd NO ne oe l S curit Biologique 222 0 cece cece cece cccecceccececceecetceeceeeeeeeeceeceeeee l Spare Parts ooi aa aaa l Chapter 2 Experimental Design cece ec kaaa 9 Sample Preparation le 9 Experimental Design 22 22 ccc ccc e cc ecc ec cccccecceccceeceeceeceeceeseeeeneees 9 FlowSight Fluorochrome Charts 200 000 0020 cece cece ccc ec ccc ceccececcceeceeceecees 11 Chapter 3 Using the FlowSight 2222200 0200 00 c aaa 13 Operating the FlowSight 2 13 FlowSight User Interface 2 0 00 o oo ccc cece ccc ccc ccccccecceeceeceeceeceeeeeeeaees 14 The Image Gallery 22 14 Image Gallery Tools 22 00 00 0 0 cece cece cece cece ec cecccceccecceeceeeceecteeeeeeeeeeeeeee 14 The Analysis Area GANAN hee Rare NN alee a nee ee 15 Chapter 3 The Instrument Control Panel 0 16 REIGNS 2 de 19 Sterilizer Cleanser and Debubbler 0 00 0 20 o cece a 19 Waste NIG ES Ara hate a ILLEGAL A LUANE naaa DY 19 Sheath Fluid LL 19 Fluid level
9. to acquire after Collect and select the pop ulation To add another population click the box 7 Select the channels to collect if desired See Display Properties below 8 Enter the file name The number in the Sequence box is appended to the file name followed by the Fif extension The sequence number increases by 1 with each successive data acquisition Files collected with BF off will be appended with noBF File names must be 256 or fewer characters in length including the path and file extension In addition file names cannot contain the following characters lt gt or 9 Browse to select an existing folder or to create a new folder in which to save the files Note do not store data on the imaging computer 10 Click the Acquire button to acquire the data if desired to collect this file 11 The data file s are automatically saved in the selected folder once the desired number of objects are collected To prematurely stop acquisition click The system prompts you to either discard the acquired data or to save the collected data in a file The acquisition can be paused and resumed by clicking L Eo Chapter 3 A Acquiring File default_1 rif All 0 25 Validating 12 Once acquisition finishes either press to load the next sample or Return return the remaining sample Note If the next sample has no nuclear dye and follows a DNA intercalating dye stained sample run Load a
10. two drops of FlowSight calibration beads Vortex first 5 Inthe ASSIST view press Start all calibrations and tests Calibration takes 12min and when all tests pass the system is ready to run for the day 6 Select load default template or experiment template from the File menu 7 Press Load and load an aliquot of a sample with each fluorochrome present Run on Low speed for best sensitivity 8 Inthe Illumination section turn on the appropriate lasers for each fluorochrome in the experiment 9 Adjust the laser power to maximize brightness and prevent saturation The 405 laser power should be adjusted to use a minimal acceptable power to prevent autofluorescence and PE excitation into the BF9 reference channel 10 Create dot plots and regions to identify the cells to collect or collect All events 11 Set the acquisition parameters including file name destination folder number of events and population s to collect 12 Choose file format either rif IDEAS fcs FACS software or both from File menu 13 Compensate data if needed Data can be recompensed in IDEAS post acqui sition A Open the compensation wizard B Load a compensation single color sample C Verify the system finds the correct channel D Set the population to save E Set the acquisition file name and destination F Press next to save the data file G Repeat steps B through F for each compensation sample Press exit and save to return to the ex
11. 5 1e6 1e7 1e4 leJ 0 163 1e4 1e5 1e6 1e7 Uncompensated Intensity MC Ch11 Intensity MC Ch11 For example changing the matrix value in the 2x2 submatrix from 0 189 to 0 10 changes the compensation as shown from the graph above properly com pensated to the graph below under compensated Compensation Coefficients a a NG Na 12 pa Uncompensated Intensity MC Ch led 1e3 0 1e3 led 1e5 1e6 le7 Uncompensated Intensity MC Ch11 ki amp S KG gt ka 1e4 1e3 0 1e3 1e4 1e5 Intensity MC Ch11 18 Repeat from step 13 for each single color compensation sample in the exper iment 19 Click Finish and save the matrix ctm when done The previous settings will be restored and the compensation matrix is now being applied to the Intensity features and images This is indicated by the com pensation button in the lower task bar Compensation 9 Note Uncompensated pixel values will still be collected You are now ready to continue collecting the rest of the experimental samples Note The experimental template is reloaded when the compensation wizard is finished If you are collecting compensation files without using the wizard be sure 28 Data Acquisition to turn the 785 scatter laser back ON brightifield ON and select the channels to acquire if desired 20 Press Load and load the next experimental sample 21 Enter the number of cells you want to acquire after Collect and select the pop ulation 22 Chan
12. 60 Hz and 1 5A Electrical hazards are present in the system particularly in the main power supply To protect against electrical shock you must connect the instrument to a properly grounded receptacle in accordance with the electrical code that is in force in your region S curite Electronique Alimentation 100 240 V altenatif 50 60 Hz 1 5A Les hazards lectrique se trouvent dans l appareil surtout pres de la source d alimentation Pour viter les choks lectriques introduire la lame le plus large de la fiche dans la borne correspondante de la prise et pousser a fond Laser safety The FlowSight is a Class 1 laser device and complies with the U S FDA Center for Devices and Radiological Health 21 CFR Chapter 1 Subchapter J No laser radiation is accessible to the user during normal instrument operation When the hood is removed interlocks on the instrument turn the lasers off The FlowSight may have the following lasers Table 2 Wavelength Maximum Power 00 413 nm 150 mW 83 493 nm 60 mW 58 562 nm 50 mW 35 647 nm 150 mw 75 795 nm 90 mW The following laser waring label appears on the interior side panels near release mechanisms Chapter 1 Caution Using controls making adjustments or performing procedures other than those specified in this manual may result in hazardous exposure to laser radiation S curit Laser L FlowSight c est une appareil au laser Classe qui se conforme a U S FDA
13. F and 785 nm illumination OFF and enable all 12 channels 26 Data Acquisition All 373 obj s i Qa Gan S 9 L Rt t Bass ED Ch01 h02 03 Ch04 Ch05 Ch06 Ch07 Ch Ch09 Ch10 Chil Chi2 Verify that the selected channel is appropriate for your sample Make any appropriate changes and click Next Chot Ch02 Chos Ch07 Ch10 16 Verify the channel for the sample loaded and click Next The compensation coef ficients are calculated and added as shown in the table Compensation is applied to the Intensity feature and images Two scatter plots are added to the work area One of Uncompensated Intensity and one of Intensity for the peak channel vs the adjacent channel is added to the work area a k ABA sa 4 Uncompensated Intensity MC_Ch12 1e3 0 1e3 Ted 1e5 1e6 le7 names o Uncompensated Intensity MC Ch11 AF647 noBF 1 nf 0 500 a e Validating 1 Load color compensation control sample _Ch12 Elapsed Time 2 Verify active fluorescent channel 3 Setup file acquisition Intensity MC Tip You may draw a region in the scatter plot to create a new population Computing coefficients 17 If desired compensation values can be adjusted by clicking on the compensation tool button in the graph Li 97 Chapter 3 DI E lai AA kd Ch12 MC_Ch12 1e3 0 1e3 Uncompensated Intensity M Intensity le4 1e4 1e3 0 1e3 ted 1e
14. FLOWSIGHT INSPIRE for FlowSight Software User s Manual september 2013 FlowSight version 100 2 195 Log in to your account at www amnis com login html to check for updates Preface Patents and Trademarks Amnis Corporation s technologies and products are protected under one or more of the following U S patents 6211955 6249341 6256096 6473176 6507391 6532061 6563583 6580504 6583865 6608680 6608682 6618140 6671044 6707551 6763149 6778263 6875973 6906792 6934408 6947128 6947136 6975400 7006710 7009651 7057732 7079708 7087877 7190832 7221457 7286719 7315357 7450229 7522758 756 695 Additional U S and corresponding foreign patent applications are pending Amnis the Amnis logo ImageStream FlowSight INSPIRE IDEAS SpeedBead FISHIS are registered or pending U S trademarks of Merck KGaA All other trademarks are acknowledged Disclaimers The screen shots presented in this manual may vary in appearance from those on your computer depending on your display settings The Amnis FlowSight cell analysis system is for research use only and not for use in diagnostic procedures Technical Assistance Amnis Corporation 645 Elliott Avenue W Suite 100 Seattle WA 98119 Phone 206 374 7000 Toll free 800 730 7147 WWW amnis com Table of Contents Table of Contents EE E AA 0 Patents and Trademarks 0 0 0 2 0c ccc e cece eee ce cece eee eccececeececeeeeeeeeee 0 DISCIAIM FS
15. ams Verify brightfield is working normally and rerun ASSIST using calibration Possible Causes Recomended Solutions Camera is not track ateral deviation of he core stream due o air or clog in the Run the purge bubbles script from the instrument drop down menu See solu tions for unstable fluidics In the Focus and Centering section adjust focus and centering left or right until the images are centered and in optimal focus The core tracking and focus tracking should not change significantly from day to day If either value changes rad ically objects may rotate due to inter actions with the sheath An off center core stream is caused by air or clogs in the fluidic system Run the purge bubbles script from the instrument drop down menu See solutions for unstable fluidics Re run the focus adjuster and frame offset calibration in ASSIST and verify it passes Autofocus and cen tering is not tracking properly Core stream posi tion is grossly off center within the flow cell due to air or clog in the fluidics Camera line rate is incorrect Excessive core stream variation due to air or clog in the fluidics Core stream is mov Allow the system to settle for 60s ing too fast for the jeconds after loading a sample Collect camera data once imagery looks good In the Focus and Centering section Autofocus is not adjust focus and centering left or right tracking properly until the images are centered and i
16. ation 56 nm Horizontal Laser Calibration 642nm Horizontal Laser Calibration 405nm Laser Power Test Airnm Laser Power Test 55 nm Laser Power Test 642m Laser Power Test 785mm Laser Power Test Alignment Test ightfield Uniformity Test Camera Noise Test Flow Core Axial Stability Test Last Run Time 1127 2073 11 55 14 AM 2417 2013 11 55 27 AM 4172013 11 56 12 AM AV A2013 11 56 36 AM 7 17 2013 11 5640 AM 7 17 2013 11 57 09 AM 7 17 2013 11 57 50 AM 2172013 11 58 33 AM FFT 72013 11 58 43 AM 7 17 2013 11 59 14 AM 2172013 11 59 50 AM 4172013 12 00 48 PM 3417 2013 12 01 00 PM 112013 12 01 06 PM 21772013 12 01 11 PM FAT 72013 12 01 17 PM 7 17 2013 12 01 22 PM 2172013 12 01 23 PM 2417 2013 1201 40 PM 7 17 2013 12 02 25 PM 2172013 12 02 23 PM 7 17 2013 12 03 03 PM 2172013 12 03 17 PM FAT 72013 12 03 31 PM 3417 2013 12 03 47 PM 21772013 12 04 33 PM 7 1 2013 12 05 13 PM 1111111 I ill Start All Calibrations and Tests Stop All Data Acquisition Data Acquisition After the FlowSight system is calibrated you are ready to acquire experiment data files The sample is loaded into the sample pump Sample is injected into the flow cell to form a single core stream that is hydrodynamically focused in front of the imaging objective Refer to the Au
17. bjects are out of focus Objects are cropped The two brightfield images are not of the same cell _ A5 Chapter 4 Images appear pixelated or larger than normal Not all 12 channels are being displayed Images have incorrect colors Intensity Fluorescence imagery appears too dim Fluorescence is too bright images have a contrasting color or appear flat One channel saturates while the others do not Scatter is too dim or bright or changes over time Large variation in brightfield intensity levels Brightfield intensity level sets incorrectly System Symptom Possible Causes Recomended Solutions Make sure a sufficient sample volume is used 50ul is recommended To clear the air bubble Run the purge bubbles script Detergents and foaming agents such as FBS can cause bubbles to form in the lines If these buffers are causing air in the system remove them from the sample and resuspend in dPBS Run the purge bubbles script Run the sterilize script followed by the startup script Load calibration beads and verify the system runs normally Filter the sample with a 7Oum nylon cell Strainer Run the sterilize script followed Clog in fluid lines by the startup script Load calibration beads and verify the system runs nor mally Clumpy and viscous samples cause cav itation in the fluidic lines and create bub Sample is too con bles Dilute the sample to 1x10 7 centrated cells ml and strain the cells through a 7Qum nylon mesh R
18. cond and without sig Calibration particles nificant clumping If the beads are diluted are not running or clumped try running a fresh tube of properly beads If the problem persists there may be a fluidics issue see the Flow rate stops or slows over time section Tests may fail if the system is reloading Calibration and or sheath or failed to set up properly Re test failure run the test by clicking in the box next to the test and pressing the start button in _ 48 Chapter 4 Troubleshooting Symptom Possible Causes Recomended Solutions the popup window If the test fails three times in a row call Amnis service Focus adjustor cal aya Nan aa baion AA Verify brightfield is working properly Frame Offset cal epee eration Failure Verify brightfield is working properly Spatial Offsets cal NAO bration failure Verify brightfield is working properly Make sure the excitation lasers are off Dark Current cal jand brightfield is blocked Completely ibration failure power down the FlowSight and power back up to re run the test Brightfield XTalk Verify brightfield is working properly and calibration failure that spatial offsets passed Verify the laser turns on and can set Horizontal Laser power properly Completely power down Calibration failure the FlowSight and power back up to re run the test Verify spatial offsets passed Verify the laser turns on and can set power properly Verify spatial offsets and fram
19. d labeling Here are a few suggestions to help design the experiment e Try to achieve at least a full log shift in fluorescence as measured by a standard flow cytometer e Use the brightest dye for the antigen with the small est copy number e The brightness of probes can be independently con trolled by changing the laser power However data quality is enhanced when the brightness levels of all probes excited off a single laser are balanced to within a log of each other Probe balancing avoids the saturation of bright stains when they are com bined with dim stains in the same sample 10 _ Chapter 2 Experimental Design FlowSight Fluorochrome Charts Ch Band nm BRIGHTFIELD Excitation Laser nm BRIGHTFIELD 85 used i Recommended dyes based on optimal excitation and detection channels are in boldface Ideal dyes with 1 2 or 3 laser systems 1 laser 488 AF488 PE PE TxRed PE Cy5 PE Cy7 SSC Ch6 2 laser 488 642 AF488 PE PE TxRed SSC Ch6 AF647 APC Cy7 3 laser 488 405 642 AF488 PE PE TxRed SSC Ch6 DAPI AF647 Band pass filter values assume 3 laser configuration s414 Chapter 3 Using the FlowSight Chapter 3 Using the FlowSight Operating the FlowSight This chapter describes the operation of the FlowSight system using the INSPIRE for FlowSight software Daily operation involves an initialization of the system and calibration using the Calibration bead reagent
20. d sensitivity for the run gt i Sensitrity LO Bk Lo Soong Hi Speed and Sensitivity are inversely related Hi Sensitrvity Lo fluidics the system 18 Reagents Reagents Sterilizer Cleanser and Debubbler These recommended reagents have been formulated to optimize the performance of the FlowSight seals valves syringes and lines The use of the recommended reagents is required for proper operation of the instrument The Sterilizer Cleanser and Debubbler reagents are used in the Sterilize and Debubble scripts Reagent Name Source Catalog terilizer WR JT9416 1 ebubbler 70 Isopropanol Millipore 1 37040 BSS 1006 B 1X Sheath PBS Ca Mg free Millipore 6506 1L 10X Rinse Deionized water Calibration FlowSight Calibration Amnis 400300 Beads Beads provided for information only other sources of the same reagent may be used Waste Fluid The two liter waste bottle holds all of the fluids that have been run through the FlowSight Add 200 ml of bleach to the empty waste tank It is recommended that the waste bottle contain 10 bleach when full Sheath Fluid Two bottles are provided one labeled Sheath to be filled with phosphate buffered saline PBS with no surfactants for running samples and one labeled Rinse to be filled with de ionized DI water for rinsing the instrument during shutdown Fluid is drawn from these bottles into the sheath and flush syrin
21. deviation of the background intensities Camera Line Number none An incremental count of objects The clock rate in KHz This is rel Camera Timer none l ative time M01 Ch01 through Enumerates changes of pixel values in the image to measure M12 Ch12 the focus quality of an image atensi MC_Ch01 through The sum of the pixel intensities in y MC_Ch12 the mask background subtracted Major Axis wor ao12 Describes the widest part of the mask M01 _Ch01 through The average pixel value within Mean Pixel M12 Ch12 the mask background subtracted Minor Axis M01 M012 Describes the narrowest part of the mask Object Number none The sequence of objects Gradient RMS 31 Chapter 3 Name Mask Channel The central tendency of the pixels Raw Centroid X none along the X Axis and Y Axis respectively The central tendency of the pixels Raw Centroid Y none along the X Axis and Y Axis respectively MC Ch071 through Raw Max Pixel MC Ch12 The largest pixel value Ki MC_Ch01 through Raw Min Pixel MC Ch12 The lowest pixel value Uncompensated Inten MC Ch01 through Se aan nels panier sity MC_Ch12 g no compensation applied 1 For a standard experiment create a scatterplot of Area versus Aspect Ratio of the brightfield channel and draw a region around the single cells A Click the scatter plot tool and choose the population All the X axis fea ture Area M01 and Y axis Feature Aspect Ratio M01 for b
22. e in a custom filename set the sequence choose the data file folder type the number of events and choose the population to collect Collect 1000 _ Desktop Sheneeidetault_1 nif Acquire ul Pause acquisition After Acquisition begins Stop acquisition After Acquisition begins Su Filename Text Type the filename Choose the beginning sequence number Navigate to the folder to save the data Collect Enter the number of events to collect of Choose the population to collect Ed Add a second population to collect Le lumination In the Illumination section you can turn Alg bo Elm pear a mi Brighifeld laser and brightfield illumination on or off hasa 00 H mw randos and set intensities All lasers have var lable power and are defined by their exci tation bandwidth bigla DD Ex mv Set Intensity 17 Chapter 3 405nm laser excitation currently set to 488nm laser excitation currently set to 642nm laser excitation currently set to ON at 150 mW of power 785nm laser excitation currently set to OFF at 5 72 mW of power This laser is for side scatter only on Brightfield illumination is shown as ON in A channels 1 and 9 C nig Intensity of the brightfield to 800 counts ma CON Focus Centering Focus and Centering can be adjusted 02 gt 02 using the right and left arrows Sa e Runs the focus pan test and sets focus automatically Adjust the speed an
23. e into the AutoSampler and press the Start button cDo o oo oo o oo n iojo jolo oo o 0 o0 0G I 00 oo oo 6660 oo 0 0 0 O 0G DDD oo oo o oo DDT o o o ooo 000 File Name 7 47 2013 A011f 7 17 2013 A02rf 7 17 2013 A03 7 17 2013 B014 7 17 2013 B02rf 7 47 2013 B031f 7 17 2013 C01 PEN 7 17 2013 C02 PEN 7 17 2013 C03 PEs 7 17 2013 D01 PE 7 17 2013 D02 PE 7 17 2013 D03 PEN 7 17 2013 E01 FITC 7 17 2013 E02 FITC 12 Check or uncheck the boxes Return samples Sterilize Shutdown Note that these boxes may be checked or unchecked while the plate is running The oper ation will apply after the current sample is finished 13 Select the wells to run they will appear in the list 38 Autosampler option Load the plate During a run 14 Click Eject Tray to extend the plate nest 15 Add at a minimum 75 vl samples to the wells of a u bottom 96 well plate and cover with Sigma Aldrich X Pierce Film XP 100 Cat 2722502 and load the plate into the autosampler 16 Place your plate in the nest with well A1 positioned at the upper left corner 17 Click Start to begin 18 The Status column will be updated for each well as it is run For each sample the instrument loads the sample performs validations acquires the data and reports the result Success or error 19 For example here is a run with a failure to detect objects in column 3 for the first 2 rows A3 and B3 failed The wells are shown in red and the error s
24. e offsets passed Verify the 785 SSC laser turns on and can set power properly Completely power down the FlowSight and power back up to re run the test Verify spatial offsets passed Verify the laser turns on and can set power properly Completely power down the FlowSight and power back up to re run the test Verify spatial offsets and frame offsets passed Brightfield align pu nor Fetes aiie Verify brightfield is working properly Brightfield uni bng ees ea s kaakibat Lee Verify brightfield is working properly Verify camera can image properly Com pletely power down the FlowSight and power back up to re run the test Flow Core Axial StajVerify the reagent buffers are full Run bility test failure the sterilize script followed by the startup 49 Retro Calibration failure Side Scatter Cal ibration failure Laser Power test failure Camera noise test failure Chapter 4 script and re run the test See solutions a ee ee Flow Core Lateral See solution above Flow core Axial sta Flow Core Position sie solution above Flow core Axial sta Focus Percentage Se solute above Flow core Axial sta Focus Uniformity Se hi above Flow core Axial sta Image Quality test Se a above Flow core Axial sta Compensation F the RE Settings section verity that 1 000 of All cells or ofa Make sure that the compensation control sample has more than 10 positive Cells are not flu events and are as
25. eferably siliconized to process samples Choice of Fluorochromes Choose fluorochromes that are excited by the lasers in your FlowSight 405 488 and 642 nm are most common Use the chart below or look online for a spectra viewer that will help you plan which dyes will work best Channel 1 9 are the most common channels for bright field SSC imagery may be placed into channel 6 if desired Dyes with an are excited by at least one laser directed to channels 1 6 and another directed to channels 7 12 For these dyes the channel that the dye will Chapter 2 appear brightest in depends on the relative laser powers used Rec ommended dyes are indicated in boldface 5 Compensation Have a sample of cells each labeled with a single color for each fluorochrome used i e FITC only cells PE only cells etc 6 Cell Aggregation Minimize aggregation problems by straining the sample through a 70 micron nylon mesh strainer or by using an anti clumping buffer such as EDTA or Accumax prior to fixation 7 Fixation If fixation is desired thoroughly fix cells with 1 formalin on ice for 20 minutes 8 Number of samples No more than 30 total for feasibility experiments Please limit the samples to the following Positive and Negative biologic controls compensation controls and experiment samples 9 Brightness of Stain and Stain Balancing Quantifying the location and dis tribution of signals in an image is a demanding task that requires optimize
26. el for acquisition Channel is not acti click on the channel column heading vated i e Ch2 and check the collected check box to save that channel Image gallery dis 7 Click on the channel column heading play is set up incor i e Ch2 and set the gain and chan rectly nel color Images have incor rect colors 55 Chapter 4 Intensity Symptom Possible Causes Recomended Solutions Fluorescence imagery appears too dim Image display set tings are set too low Increase the image display gain and or change to log in the appro priate camera channel Sample did not Look at the sample with a fluorescent label well microscope Insufficient illu mination Core stream posi tion is grossly off center within the flow cell due to air or clog in the fluid ics Excitation laser is misaligned Fluorescence is too bright images have a contrasting color or appear flat Image display set tings are set too high Instrument sen sitivity is set too high Turn the appropriate lasers on Set the laser powers to maximum and decrease them to prevent pixel sat uration If the probing protocol results in dim staining sensitivity of the instrument can be increased by changing the fluidics speed to Lo Hi sensitivity mode Run the purge bubbles script from the instrument drop down menu See solu tions for unstable fluidics Run calibration particles on the Flow Sight Load the default te
27. ensities the X axis is done using the green limit lines The range of pixel intensities will from the camera is 0 4095 The limits of the graph enable you to use the full dynamic range of the display to map the pixel intensities of the image Note Changing the display mapping does not change the data They are for display purposes only 30 Setting up the work area Setting up the work area Create scatterplots or histograms with the tools in the analysis area The quantitative features that are available to graph the data are listed in the table below Features are algorithms that are applied to a specific region of interest of the image defined by a segmentation mask and image pixel values defined by the channel image Segmentation masks are named M01 through M012 indicating intensities above background in the respective channel MC is the combination of all of the individual channel masks Feature names are described by the Feature Mask Image For example Intensity M02 Ch02 indicates the sum of all channel 2 pixel intensities in Mask M02 Note See the IDEAS User Manual for more information on features and graphing The tools functionality is the same Features available for FlowSight Name Mask Channel Area Mormorznc The size ofthe mask in square microns The ratio of the Minor Axis divided G Ue by the Major Axis Bkgd Mean Ch01 Ch12 The average intensity of the cam era background Bkgd StdDev Ch01 Ch12 The standard
28. eral categories of parameters that may be chosen as a group or individually Check or uncheck the desired parameters The user can also define custom parameters Expand the category to see the individ ual parameters To delete a custom parameter select it and use the delete key Click OK when done 6 Batching of the data into IDEAS may be done if a compensation matrix and tem plate exists for the experiment Add or Remove Columns a l Standard Parameters 3 b W Date F Wel Output File Path NW Max Acquisition Time min 7 INSPIRE Template W Error Notification Email iv Minimum Volume ul W Validate Sample Commenits W Batch Dose El Time EE Custom Parameters 7 Minimum Volume ul EH mination Parameters 3 5 T BF Channel 405 Power 405 SSC Blocker 488 Power 488 SSC Blocker 561 Power 561 SSC Blocker 594 Power 594 SSC Blocker 642 Power 64 SSC Blocker 785 Power C785 SSC Blocker El Imaging Parameters E Binning El Magnification ILA LI Enter custom column name here Add Custom Column Delete OK Canoe 7 Select wells to define by clicking a individually or Ctrl click shift click for multi select b rows or columns c color d the Select Defined button or e All Note Selecting and defining wells with shared parametersfirst and then refining the definition for sets of wells makes it easier to organize the definition 8 You can ed
29. ge pumps The sheath pump helps to control the speed of the core stream and the size of the core stream diameter The flush pump is used to clean and flush the system Fluid level warnings Sensors behind the bottles trigger a pop up window to alert the user that the volumes are low or the waste volume is high The triggers are set such that any script currently running can finish Do not remove a bottle during the script Once the script has finished remove the bottle and fill the appropriate solution Waste can be emptied at any time Dispose of the waste in accordance with local regulations 19 Chapter 3 The left window below is indicating that the Sterilizer solution is low and needs to be filled The right window is indicating all bottles are currently OK Daily Startup and Calibration Daily Startup and Calibration This section describes how to prepare the FlowSight for use The FlowSight can be left on with INSPIRE launched but the following instructions also describe how to turn on the FlowSight if the power is off Turning on the FlowSight 1 Turn on both computers The large Linux based computer runs the imaging and the small Windows computer runs the user interface Toggle the power switch on the right lower panel of the instrument from OFF to ON Log on with the user name Amnis and password IS 100 Launch the FlowSight software using the shortcut on the desktop after the com puters and instrument have star
30. ge the file name for the sample 23 The number in the Sequence box is appended to the file name followed by the rif extension The sequence number increases by 1 with each successive data acquisition 24 Click Acquire to collect the file when the cells are running smoothly 25 Repeat for each sample 26 When finished running samplesfor the day press Shutdown 29 Chapter 3 Setting the Image Display Properties 1 Click on the settings tool w or a channel name to open the window 2 Here you may Select the color for image display of each channel Select the color for displaying saturated pixels Set the display intensity mapping Enable channels to collect Run the display wizard which helps to map the display intensities Select Channel Ch01 Enable Minimum Pixel Intensity 0 Maximum Pixel Intensity 823 Channel Names Colors Cho 255 CCC DC eee Ch02 v Ch03 Ch04 Ch05 Ch06 Ch07 Ch08 Ch09 Ch10 Ch11 Ch12 R Display Wizard Automatic Manual Image Display Mapping X Axis Scale Set to Default Full Scale Set Range to Pixel Data ON OB D EE RE S S S 8 Autoscale Set Linear Curve Reset to Default Colors Saturation Color Reset All Settings Reset to White Enable All Channels Note Mapping the Display Intensity settings on the graph value of the display the Y Axis to the range of pixel int
31. idics 46 Uncompensated Intensity 32 User interface 14 Waste fluid 19 63
32. iles 25 Debubbler 19 59 Chapter 4 DNA dye cleaning 26 Experimental Design 9 Features 31 Area 24 Intensity 24 Max Gradient Intensity 24 Mean Pixel 24 Raw Max Pixel 24 Raw Min Pixel 24 Saturation Count 25 Saturation Percent 25 Fixing cells 10 Fluorochrome Chart 11 Fluorochrome choice 9 11 Fluorochromes 9 Gradient RMS 31 Graphs features 24 Image Display properties 30 60 Image Gallery 14 tools 14 Instrument Control Panel 16 Intensity 31 L Laser adjustments 24 M Major Axis 31 Mean Pixel 31 Minor Axis 31 O Object Number 31 Q Quick Start Guide 34 R Raw Centroid X 32 Raw Centroid Y 32 Raw Max Pixel 32 Raw Min Pixel 32 Reagents 19 Rinse 19 61 Index Chapter 4 Safety 1 biological 7 electrical 5 laser 5 Sample buffer compatability 23 order 23 Sample preparation 9 Saturation color 30 setting sensitivity 23 Setting up the work area 31 Sheath 19 Sheath fluid 19 Startup 21 Sterilizer 19 Symbols explanation 4 Troubleshooting 45 ASSIST failure 48 brightfield 57 58 channels displayed 55 color 55 compensation 50 sB Index cross contamination 48 erratic acquisition rate 53 event rate slows over time 47 file collection 52 fluid level indicator 48 focus 54 images 53 55 56 imaging 54 INSPIRE appears to freeze 50 launching INSPIRE 51 low event rate 47 plotupdating 51 scatter 57 sluggish fluidics 47 stain balancing 57 unstable flu
33. isition and if enough of the images are cropped the event rate can 47 Chapter 4 Symptom Possible Causes Recomended Solutions appear lower than normal Normally this is due to air in the system Run the purge bubbles script from the instrument drop down menu See solutions for unstable fluidics Turn the appropriate lasers on Set the Insufficient illu 785 SSC laser to 40mw Set the laser mination powers to maximum and decrease them to prevent pixel saturation DNA dyes must be thoroughly flushed from the sample lines to prevent residual dye from labeling subsequent samples Load a sample of 10 bleach followed by a PBS wash to remove all traces of the DNA dye in the instrument or run the sterilize script 30min Cells from the pre This suggests a minor clog Load a sam ple of 10 bleach followed by aPBS DNA dye from pre vious sample is tamination from labeling current previous samples cells or run the sterilize script 30min Tank has moved Open the buffer compartment and move away from the sen the tank closer to the sensor until the fluid sor level indicator is correct Power down and power up the instru Sensor is broken ment if this does not fix the problem call Amnis service Erroneous fluid level indicator SUNE ME wil Incorrect template Go to the file drop down and select load loaded default template Re run ASSIST The particles must be running gt 1 000 events per se
34. it values for some of the Custom and many of the Standard param eters You can do this for all selected wells or for individual wells For example if _ 37 Chapter 3 you want to collect with Max Acquisition Time 10 minutes for the selected wells type 10 in the Apply to selected box below the Max Acquisition Time heading If you want to only apply this to a single well type this value in the box cor responding to that well Below is an example of a Well Plate Definition using sev eral parameters and showing only defined wells FEROS I ERLRGGESL 9 Highly recommended select Error notification Email from the list of Standard parameters and type in the user email address in the Apply to Selected box 10 When done click Save Start the autosampler 11 Click Start to run the plate The Auto Sampler Unattended Operation window opens with the Plate Definition you just saved If you wish to choose a different Definition browse for it by clicking on the folder icon If you want to edit the Plate Definition click Edit This Plate and you will be taken to the Well Plate Definition window Uattended Ope asie Al Eject Tray P Start Plate Definition Plate Settings My Plate Definition EZ Edit Return samples F Sterilize Shutdown An 1 2 3 4 5 6 7 8 9 10 11 12 Well Definition A e e e e o O O O O 6 Select the defined wells that you wish to run 3 e e e o 0 O O O Load the plat
35. make sure the lasers The INSPIRE appli jand brightfield lamp are turned on and cation has crashed then re establish the core stream If the application does not start use the Win dows Task Manager to end the INSPIRE task again Shut the Flow Sight computer down from the Start menu Then turn on the instrument as launch not responding for FlowSight and press end task Wait 60 seconds and try restarting INSPIRE Shut down the computer and power off the instrument Verify all computers are between the com off Power on the instrument and then puters and instru the computer wait 5 min and try launch ment ing INSPIRE Plots fail to update or update resources are Close all third party software slowly being over used 51 Chapter 4 Symptom Possible Causes Recomended Solutions Too many plots in For optimal plot update rates limit the the template number of plots to 15 Right click on the plot select graph prop erties and change the selected pop Parent population has no qualifying ulation to all or a population that has events qualifying events Plots are scaled In the plot tool bar press the mag incorrectly nifying glass and rescale the plot Make sure there are events going into the collection region by viewing that region in the image gallery and updat ing the acquisition collection population appropriately Verify the cell concentration is appro priate 1x1017 cells ml is ideal Verify the c
36. ment est de niveau L1 Spare Parts The instrument contains no serviceable parts Only Amnis trained technicians are allowed to repair maintain and set up the alignment of the laser beams Chapter 2 Experimental Design Chapter 2 Experimental Design Sample Preparation Experimental Design The FlowSight system can quantify the intensity specific location and distribution of signals within tens of thousands of cells per sample The system can perform most standard flow cytometric assays and can also leverage the technology s imaging capabilities to discriminate image based changes within individual cells and cell populations 1 Choice of Cell Type The cell particle size should be less than 60 microns in diameter Basic example QI example THP 1 20um diameter THP 1 Briahtfield Fitc Drag5 Briahtfield Fitc Draa5 1289 Blood Cell 8um diameter Blood Cell Briahtfield Fitc DAPI 15614 s Briahtfield Fitc DAPI 5675 2 Final Sample Concentration and Volume The recommendation is at least 1 million cells in 50 ul 2x10 cells ml in PBS 2 FBS in a 1 5ml siliconized microcentrifuge tube Protocols In general any established labeling protocol used for flow cytom etry will work with the FlowSight see Current Protocols in Cytometry for gen eral labeling techniques Stain cells on ice in the presence of azide when possible to reduce non specific capping of antibody Use polypropylene tubes pr
37. mplate Open ASSIST re run the laser alignment calibration for the appropriate laser line and verify it passes Decrease the image display gain and change to linear in the appropriate camera channel Decrease the excitation laser power to prevent pixel saturation Saturation is indicated in the image gallery by pix els colored in a contrasting color gen erally red or white The sheath syringe Load sheath then go to the instrument is empty drop down and run prime There is a clog or Run the Purge Bubbles script from the Set the brightfield intensity to 800 counts by pressing Set Intensity Chapter 4 Troubleshooting Symptom Possible Causes Recomended Solutions air bubble in the sysjinstrument drop down menu See solu tem tions for unstable fluidics The best instrument setup maximizes the dynamic range of fluorescence sig One channel sat Instrument sen nal while at the same time avoiding urates while the sitivity is not opti image pixel saturation which cannot others do not be compensated In general decreas ing the laser powers until no pixels sat urate Reduce the concentration of the stain that produces the saturating signal so that all probes can be simultaneously imaged without excessive saturation some DNA dyes are required to run with the sample to stain properly how ever if too much dye is in solution it can cause the core stream to flu cells can be imaged properly Typ ically the
38. mplate for future use Tem plate file names are appended with the suffix ist They are saved in the INSPIRE Data folder Load Default Template Loads factory settings Generate RIF file Check to save a Raw Image File during acquisition Generate FCS file Check to save a Flow Cytometry Standard file during acquisition Exit and Shutdown Instrument Turns off the instrument control system and exits INSPIRE Note this does not sterilize the system Exit Exits INSPIRE 2 Instrumentmenu Run the ImageStream camera and instrument specific fluidic scripts automated fluidic routines 41 Chapter 3 Start Camera Calibrate with ASSIST CT Cho Ch CHE pawa Load 5heath Load Flush Syringe Prime Purge Bubbles Purge Sample Load Line View Tank Levels 5 SericeScripts O Test 5cripts Options Interlock Override Change Password Enable Advanced Configuration Show Error Tab Usage Monitor Start Camera Turns on imaging if it has stopped Calibrate with ASSIST Opens the Calibrations and Tests window Load Sheath Fills the sheath syringe with sheath fluid and an air bubble that facilitates stable flow Load Flush Syringe Fills the flush syringe with sheath fluid Prime Rapidly pushes a small volume of sample into the flow cell Purge Bubbles Removes air bubbles from the flow cell by filling the flow cell with air then filling the sheath line and pump with debubbler and ri
39. n optimal focus Run the purge bubbles script from the instrument drop down menu See solu tions for unstable fluidics _ 54 Chapter 4 Troubleshooting Symptom Possible Causes Recomended Solutions Lateral deviation of the core stream due to air or clog in the Run the purge bubbles script from the instrument drop down menu See solu tions for unstable fluidics In the Focus and Centering section adjust focus and centering left or right until the images are centered and in optimal focus Run calibration particles on the Flow Sight Load the default template and Frame offset is incorjverify brightfield is in channel 1 and 9 at 800 counts of background Open ASSIST re run the frame offset cal ibration routine and verify it passes Call service and verify that the illu mination pathways are in proper align ment Run calibration particles on the Flow Sight Load the default template and Cross correlation is verify brightfield is in channel 1 and 9 incorrect at 800 counts of background Open ASSIST re run the cross correlation utility and verify it passes tering is not tracking properly The two brightfield images are not of the same cell Illumination is grossly misaligned Image gallery zoom Use the magnifying glass to zoom is active out and restore the native image size Image gallery zoom Use the magnifying glass to zoom is active out and restore the native image size To activate a chann
40. nsing the flow cell The sheath syringe is then refilled with sheath and the bubble trap lines and flow cell are filled with sheath Purge Sample Load Line Cleans out the sample load line View Tank Levels Opens the fluid level window Service Scripts For field service personnel only Usage Monitor is avail able to find the information on scripts that have run Options 3 Analysis menu Access the Feature Population and Region Managers Func tionality is the same as for IDEAS Refer to the IDEAS user manual for more information Analysis Features Populations Regions T Wizards 42 Menus Features Opens the Feature Manager Features can be renamed or new combined features can be created Populations Opens the Populations Manager View edit or delete pop ulations Regions Opens the Regions Manager View edit or delete regions Wizards Opens the list of Wizards including the compensation wizard Layout menu Layout Vertical Honzontal Auto resize Analysis Area Vertical View the image gallery and analysis area side by side Horizontal View the image gallery and analysis area top and bottom Auto resize Analysis Area Adjusts the layout to accomodate graphs and images when the image gallery changes size Compensation menu View edit or create a new compensation matrix J Compensation Li Create Matri
41. omputer hard drive has suf ficient room to save the data file To do this go to Start Computer right click on properties and a pie chart showing how much disk space is available is dis played Backup and delete data to free up disk space Some samples have high con centrations and acquire faster than the Data file fails to No events qualify collect for the region Data file collected rapidly display rate Check the destination folder and see if the raw data was col lected Collecting data over a downed network or changing the name of the destination folder will cause FlowSight to lose the data directory Verify the data des tination folder is accessible using the browse button in the Acquisition Set tings section No rif or fcs file Go to the file drop down menu and was created check Generate rif and or fcs file File directory was lost 52 Chapter 4 Troubleshooting Image Symptom Possible Causes Recomended Solutions If the camera is Click Stop to stop the camera and a running ne click Run Setup UU magingis paused Displayed region is the cell view area select the all pop incorrect ulation Turn the appropriate lasers on Setthe Insufficient illu 785 SSC laser to 40mw Set the laser mination powers to maximum and decrease them to prevent pixel saturation Make sure the brightfield lamp is turned on andclick Set Intensity Core stream is out Manually find the core stream In
42. or Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements EN 61010 1 2001 2 edition AUTHORIZED REPRESENTATVE Izasa SA Aragon 90 08015 Barcelona Spam VAT ESA28114742 Contact Jose Mana Alonso 34 916 630550 TEST FACILITY F Squared Laboratories Northwest EMCI 26501 Ridge Road 22913 NW Evergreen Parkway ste 400 Damascus MD 20272 USA Hillsboro OR 97124 USA The Cellular Analyzer Model FlowSight is in effective conformance to the Directives and Standards referenced above Name Richard Esposito Title Quality Assurance Officer Authonzed by Date 27 July 2011 Chapter 1 Explanation of symbols Explanation of symbols Risk of expo sure to trans Waste tank missible biological dis ease Risk of injury Power supply cover by electric shock Protective POWeroUppiy earth ground DANGER Risk of expo Laser radiation when open sure to haz and interlocks defeated Inside rear frame panel d AVOID EXPOSURE TO BEAM ardous laser radiation interior side panels near Risk of expo release mechanisms sure to haz and next to hood inter lardous laser locks radiation No laser radi ation is acces sible to the user during normal instru ment oper ation On the back of the instru ment Chapter 1 General Information and Safety Electrical safety Equipment ratings The FlowSight is rated to the following specifications 100 240 VAC 50
43. panning three pixels in an image This feature measures image contrast or focus quality e Intensity The integrated intensity of the entire object image the sum of all pixel intensities in an image background subtracted e Mean Pixel The average pixel intensity in an image background subtracted e Raw Max Pixel The intensity value of the brightest pixel in an image e Raw Min Pixel The intensity value of the dimmest pixel in an image _ 24 Data Acquisition e Saturation Count The number of pixels in an image that have an intensity value of 4096 e Saturation Percent The percentage of pixels in an image that have an inten sity value of 4096 Collecting and saving the data files Once the sample is running and the FlowSight is properly set up you are ready to acquire the data as a raw image file rif This file contains uncompensated pixel data along with instrument settings and ASSIST information in a modified TIFF format The file includes only those objects defined by the population selected in the acquisition section As an option a compensation matrix may be applied to the data as it is collected by either going through the compensation wizard to create the matrix or choosing a matrix from the File menu The setup can be saved as a template file ist for future experiments Filename Seq 2 defaut E CP Collect 1000 H of A Desktop Sheneeidetault_1 nif Acquire 6 Enter the number of cells you want
44. per iment 14 Continue collecting all experiment files using consistent instrument settings n general brightfield will be in channels 1 and 9 SSC 40mw in channel 6 and the cells to collect R 1 using brightfiled area vs aspect ratio to identify single cells 15 Save an experiment template by selecting Save Template from the File menu 16 Shut the system off by pressing the Shutdown button The system sterilizes in 740 min 34 Quick Start Guide to FlowSight Operation HI o O pa CE a 35 Chapter 3 Autosampler option The autosampler enables unattended operation of samples in 96 well plates loaded into the FlowSight Prior to running the plate a plate definition is created that assigns instrument settings to the wells names to the output files and parameters to include in a well plate report While the plate is running the user may be notified of any errors encountered via email The instrument can also sterilize at the completion of the plate This document describes the steps involved in operation the FlowSight autosampler Workflow 5 Startup the instrument and calibrate as described under normal operations Create Instrument Settings Template s ist to be used for each well on your plate To do this run an experimental sample manually with all of the flu orescence dyes to be used in the experiment see INSPIRE Setup Quick Start Guide Save each relevant template Create a Well Plate
45. ponsibility of the end user Follow all local state and federal biohazard handling regulations for disposal of the contents of the waste reservoir Prevent waste reservoir overflow by emptying the container when the waste indicator indicates that it is full Run the instruments sterilize routine after each day s use Note that this procedure has not been proven to result in microbial sterility S curit Biologique Biorisques L FlowSight est valu un niveau de s curit biologique L1 Ne pas acqu rir ou vider des chantillons contenant des agents infectieux sans les avoir inactives Il est recommande que les echantillons soient fixes dans du paraformaldehyde 2 pendant au moins 10 minutes avant d acquerir des chantillons avec I FlowSight L utilisation le confinement et l limination des mat riels biologiques dangereux sont tenus d tre en conformit avec les normes de s curit relatives au laboratoire et de la directive 93 95 E et restent sous la responsabilit de l utilisateur Respectez la r glementation en vigueur pour le traitement et l limination des d chets dans des r servoirs pr vus cet effet Pr venir l accumulation des d chets en vidant le r servoir lorsque l indicateur indique qu il est plein St riliser les instruments de routine apr s chaque journ e d utilisation Notez que cette proc dure ne garantit pas la st rilit vis vis des microbes Le Niveau de s curit biologique pour l instru
46. pty or the waste tank full during a run an alert will be sent to the email entered in the well plate def inition Acquisition will pause until the user intervenes e fan error occurs on a well the sample is returned an alert is sent to the email address entered in the well plate definition and the autosampler moves on to the next well e If the same error occurs on three consecutive wells the autosampler aborts the plate and sterilizes the instrument if the Sterilize after running plate box is checked 39 Chapter 3 Report 20 A report will be saved to the folder designated in the Output File Path of the plate definition at the end of the run either when it was stopped manually or completed the entire plate 40 Menus Menus The menu bar is located in the upper left portion of the INSPIRE screen It consists of the following drop down menus This section describes the functions that are assessable from the menus 1 File menu Load and save instrument setup templates A template contains instrument settings that can be predefined and loaded to simplify the instrument setup process File E or le New Template Load Template save Template Load Default Template Generate RIF file Generate FCS file Exit and Shutdown Instrument Exit New Template Removes all graphs populations and regions Load Template Browse for and open saved templates Save Template Save your settings as a te
47. rightfield in channel 1 B Click the polygon region tool and click on the graph to create the region A population is created and the population becomes available in the list at the top of the image gallery C Single cells will have an aspect ratio around 1 The Area of the debris is small and doublets or aggregates will be larger In this example R1 is drawn around the single cells and R2 is debris amp DE o in Pg peci Ratia MOT ra ka 600 900 1 283 Area M01 30 Setting up the work area 2 Confirm your gating by viewing the populations created by gates in the image gallery 3 Create scatterplots and histograms as required Example template for 4 color experiment amp C Li amp lt 0 E Choa Aspect Ratio MAD mensi MC LEJ jad Intensity MC Cho Li amp QD 8 j mi MU onl ik Intengdy ME CAT intents Tes 185 ke ie ied Irene MAC Chi riens y MC Cho 3 Normalized Frequency fal Lal Iniensiy MC CH07 Refer to the IDEAS User Manual for more detailed information on graphing tools moving regions or panels or managing populations mi BI Chapter 3 Quick Start Guide to FlowSight Operation Power up the system and launch the FlowSight application 2 Check to be sure the buffer containers are full and the waste tank is empty 3 Select Startup and the instrument will flush the system and load sheath in 12min 4 Press Load and load
48. rochrome has Raw Max Pixel Intensities between 100 03 Chapter 3 and 4000 counts as measured in graphs and there is no saturation The default saturation color can be changed by clicking Channels in the Acquisition section In the examples below the objects are not saturating in channels 2 5 or 7 but saturating in channel 11 indicated by the events lining up at 4095 counts A lou D E Gs Ga Pi i a Ww a F E3 o Ci m a Dr a io Raw Max Pixel MC Ch11 183 2e3 323 200 400 600 800 Raw Max Pixel MC Ch0 Raw Max Pixel MG Ch07 saa Illumination mW Brighifield mw land3 B47 g DD m Set Intensity 785 3 1800 mu 3 Select Brightfield channels Default is Channels 1 and 9 Click Set Intensity 4 Set the display properties if needed See Setting the Image Display Properties 5 Create graphs to gate on cells of interest Recommended Scatterplot of Area versus Aspect Ratio of brightfield to gate on cells and eliminate debris Scatterplots or histograms of Raw Max Pixel for the channels used in the experiment for setting laser power Scatterplots or his tograms of Intensity for the channels used in the experiment To identify objects for inclusion in or exclusion from the acquiring data file the fol lowing features in any channel are available e Area The number of pixels in an image reported in square microns e Max Gradient Intensity The value of the largest slope s
49. solution of 10 bleach and then PBS to ensure that residual dye does not stain the subsequent samples Collecting compensation files The compensation samples are collected using the compensation wizard or may be collected manually by turning brightfield and the 785 scatter laser OFF and enabling all channels In either case the experimental data files may be compensated in IDEAS during analysis Collect compensation files with the same laser power settings as the experimental samples 13 To use the compensation wizard click to open the wizard list and select Compensation Compensation Load the single color compensation control sample If a sample is already running click Next or Exit to finish 1 Load color compensation control sample Sample 2 Verify active fluorescent channel 3 Setup file acquisition Load Return Volume 0 0uL Time 0 00 All 5 obj s Tip 1 Turn ON lasers used for the experiment Tip 2 Once the sample is loaded verify that objects appear in at least _ _ one fluorescent channel Tip 3 Run the DNA dyes last Tip 4 Samples will be returned automatically at the next sample load 14 Click Load Return or Compensation Sample Load depending on your inter face to run the first compensation single color sample Note if sample is already running prior to launching the wizard go directly to the next step 15 Click Next when the sample is running The wizard will turn the B
50. tatus is dis played An alert email was sent as entered in the plate definition BE a Eject Tray O Start Plate Definition Plate Settings My Plate Definition gai Return samples F Sterilize Shutdown aja 2 3 4 5 6 7 8 9 10 11 12 Well Definition alo o LI o 0 O O O O 6 Select the defined wells that you wish to run ma on 00000000 Load the plate into the AutoSampler and press the Start button closes 0 1616160 00000000 cols ele o 000000 66 60 o 0000000 s D D Djeo o 0 o o ooo HD D Dleooo o o o oe na mapang Fle Name Status 0000 21 ADI_mlo amni com_lug_Ptl_Mag40x ni Success 001004 AN nlo amnis com lug P2 Mag4O0X noBF nil Success No File Collected Eror Object rate too slow rate 0 00 0011 BO1_mlo amnis com_10ug_Pti_Mag 40X ni Success 001022 BO2_inlo amnis com_10ug_Pt2_Mag 40 lt n0BF cf Success No File Collected Error Object rate too slow rate 0 BER nes com_1 Vehdahng baught held ntenally C02 mloG amns com 10D0ug P12 MAGNIFICATION Ide C03 mloG amns com 10Dug PtI MAGNIFICATION Ide DOT imlo amres com lug Pil BMAGNIFICATION nf Ide D02_info anmns com_lug_Pt2_BMAGNIFICATION ni Ide D3 DOG into amnres com lug PtI MAGNIFICATION ni Ide e You may stop the plate at any time by clicking the Stop button This does not initiate sterilize even if the Sterilize after running plate box is checked e Should the sheath tank or beads reservoir become em
51. ted Preparing to run calibration 4 5 Fill the rinse bottle with deionized water and the sheath bottle with PBS Empty the waste tank Push on the quick disconnect buttons to remove the tub ing from the waste tank Add 200 ml of bleach to the two liter waste bottle The final bleach concentration for a full waste tank should be 1090for sterilization Click Startup This script fills the system with sheath and flushes out all of the storage rinse that was in the system 12 minutes Click Load and place a tube containing 2 drops of FlowSight Calibration reagent on the uptake port Vortex the reagent before use Click Start All Calibrations and Tests at the bottom of the ASSIST view The calibrations and tests will run and the results saved to the database Note Instrument calibrations may also be run individually by select ing a particular procedure under Calibrations or Tests Next to each is a green or red rectangle If the procedure fails it turns red If a procedure fails repeat it If it fails twice see the troubleshooting chapter 4 or call your Amnis Field Service Representative When the calibrations and tests have all passed close the ASSIST window This window can be re opened under the Instrument menu i OA x Chapter 3 Focus Adjustor Calibration Spatial Offsets Calibration Dark Current Calibration Brighifiedd Talk Coefficient Calibration 405nm Horizontal Laser Calibration 488nm Horizontal Laser Calibr
52. the side the objective s focus and centering section move field of view core track left or right to find the core Computer resources se all third party software are being over used Imaging and acqui Make sure the sample concentration is sition rate is Sample con between 107 and 108 cells ml Lower erratic or appears centration is low concentrations can be used but this frozen will decrease the cells second Region being In the cell view area select the all pop viewed has few or lulation or readjust regions to include no cells more cells Turn the appropriate lasers on Set the Insufficient illu 785 SSC laser to 40mw Set the laser mination powers to maximum and decrease them to prevent pixel saturation Make sure the brightfield lamp is turned on andclick Set Intensity The process of object detection can safely handle up to 4000 objects per second The maximum sample con The sample is too concentrated centration is 4 5x108 cells per mL with the recommended concentration 1 10 x107 cells per ml To decrease the event rate dilute the sample Pp The sample has an Use a region to eliminate the debris nce Chapter 4 Symptom Objects are not cen tered in the chan nel Objects are rotat ing in the core stream Objects are out of excessive amount offfrom the data file or prepare a fresh debris sample Computer resources are being overused Exit all third party progr
53. tosampler upgrade section below for using unattended operation Refer to the Sample Preparation Guide or Chapter 2 for experimental set up recommendations Use compatible sample solutions from the table below Cells in PBS run with water sheath will swell Sample order Samples from an experiment are typically run in the following order e Experimental sample with the brightest stains to set the sensitivity for the run e Single color DNA dye control NO BF or SSC to ensure correct dye con centration e 10 bleach to wash out DNA dye followed by PBS e Single color fluorescence compensation samples no DNA dye NO BF or SSC all channels enabled e The rest of the experimental samples with DNA dye if applicable Loading and running the sample 1 Press Load and load an aliquot of the brightest sample in the experiment that fluoresces with each fluorochrome used It is critical that you run this sample first to establish the instrument settings DO NOT change laser settings for the exper iment once established on this sample Note Application specific instrument settings can be saved in a template and used to facilitate instrument setup but it is recommended that you verify the appropriateness of the settings for the specific experimental run 2 Turn on each laser used in the experiment by clicking on the wavelength Create scatter plots of Raw Max Pixel for the channels used in the experiment Set the laser powers so each fluo
54. ts were designed to provide reasonable protection against harmful interference when the equipment is used in a commercial environment This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instruction manual can cause harmful interference to radio communications The operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at the user s own expense Chapter 1 General Information and Safety Declaration of Conformity DECLARATION OF CONFORMITY IN ACCORDANCE TO ISOTEC GUIDE 22 Cellular Analyzer MANUFACTURER Arnis Corporation 2303 Thind Avenue Suite 210 Seattle WA 98121 Phone 206 576 6865 MODEL NUMBER FlowSight REPORT AMNI0005 and F2LOMS37 DIRECTIVES EMC Directive 2004 108 EC amp Low Voltage Directive 2006 95 EC STANDARDS Electrical Equipment for Measurement Control and Laboratory Use EMC Requirements Part 1 General Requirements EN 61326 12006 edition Industrial Scientific and Medical Equipment Radio Frequency Disturbance Characteristics Limits and Methods of Measurement CISPR 11 2009 edition Information Technology Equipment Radio Disturbance Characteristics Limits and Methods of Measurement EN 55022 1998 CISPR 22 1997 edition Electromagnetic Compatibility EMC Part 3 2 Limits Limits for Harmonic and Emissions Equipment
55. un the purge bub bles script Air bubbles in fluid lines 46 Chapter 4 Troubleshooting Symptom Possible Causes Recomended Solutions Verify the sheath is dPBS De gas the sheath as appropriate Third party sheath buffers cannot be used on the FlowSight With the system powered down look for leaking sheath Verify the fluid lines mount snuggly into position Call Amnis service The sheath syringe should contain 2 4 mL of air to buffer the movement of the pump s microstepper motor If too little air is present run the start up script With the system powered down look for Fluid lines are leak leaking sheath Verify the fluid lines ing mount snuggly into position Call Amnis service Cells settle in the lines after 45 60 min utes of running resulting in a drop in cell event rate Stop and save the acquisition Return the remaining sample restore the sample volume to 30ul and re load the sample to continue acquisition Data can then be appended together in IDEAS Air buffer in the sheath syringe is Event rate slows Cells have settled in the lines Fluid lines are leak leaking sheath Verify the fluid lines ing mount snuggly into position Call Amnis service Make sure the sample concentration is Sample con between 10 and 108 cells ml Lower con centration is low centrations can be used but this will decrease the cells second Cropped images will be eliminated from Core is off center data acqu
56. ve the plug from the socket outlet Transportation The FlowSight relies on many delicate alignments for proper operation The machine may be moved only by an Amnis representative Chapter 1 Cleaning Clean spills on the instrument with a mild detergent Using gloves clean the sample portal and sample elevator with a 10 bleach solution Dispose of waste using proper precautions and in accordance with local regulations Preventative maintenance The FlowSight contains no serviceable parts Only Amnis trained technicians are allowed to align the laser beams or otherwise repair or maintain the instrument The instrument fluidic system is automatically sterilized after each day s use This reduces the occurrence of clogging Tubing and valves are replaced by Amnis service personnel as part of a routine preventive maintenance schedule Access to moving parts The movement of mechanical parts within the instrument can cause injury to fingers and hands Access to moving parts under the hood of the FlowSight is intended only for Amnis service personnel Protection impairment Using controls or making adjustments other than those specified in this manual can result in hazardous exposure to laser radiation in exposure to biohazards or in injury from the mechanical or electrical components FCC compliance This equipment has been tested and found to comply with the limits fora Class A digital device pursuant to part 15 of the FCC rules These limi
57. w Enlarges the imagery 14 Chapter 3 Using the FlowSight Tools to measure pixel intensity of displayed images Clicking on a channel name will also open the set tings tool The Analysis Area Graphs are displayed in the analysis area during setup or acquisition Regions can be drawn on the graphs to create populations WE RHittd Ars A A Name Description O Z gt Reset ___Refeshestne gaps win incoming daa Histogram creasa rision inter Reset cursor to pointer ine region Draw a line region on a histogram P Rectangle region Draw a rectangular region on a scatterplot Oval region Draw an oval region on a scatterplot LI A I 15 Chapter 3 The Instrument Control Panel The instrument control panel provides tools to control instrument operation data acquisition and status Volume 1526 ul Time 177103 All 180 oby s Filename Seg adat E Bf Collect 1000 of A FF DeskiopiShereeldefault 1 rif Acquire 458 60 0 mw Brightfield G e F5 aw Set Intensity part of EMD Millipore Chapter 3 Using the FlowSight In the Sample section you can load a sample or return a sample Sample volume and time remaining is displayed and the rate of the selected population when a sample is running Volume 1528 ul Time 127103 All 180 obis File Acquisition Filename ond In the Acquisition Settings section you defaut ly lean typ
58. warnings 2 02 c cccceceeccececceccecceccecceececceceeceeeceeeeeeee 19 Daily Startup and Calibration cece la aaa 21 Turning on the FlowSight 22000 0 0 la 21 Preparing to run calibration 2 2 0 0 0 0 aaa 21 Data ACQUISITION a 23 Sample order aaao aoaaa anaana aaao ao ooo 00a aoaaa Aaaa AALL DLLD DLLD LALALA ARALL LL 2 nnani 23 Loading and running the sample 0 0 0000 0 0 0 lll aaa 23 Collecting and saving the data files U 0 000 0 00 0 00 25 Collecting compensation files a 26 Setting the Image Display Properties u 0 00 0 0 a 30 30 Setting up the work area 00 ll la aaa 31 Create scatterplots or histograms with the tools in the analysis area 31 Example template for 4 color experiment 0 0 ccc ccc cee eeceecceeceees 33 Quick Start Guide to FlowSight Operation 0 000 00 0 eee 34 Autosampler option 2 2 0 0 0 occ cece ccc cc cee naaa aaa DDL aaan oonan aaan 36 MN OU OM sey hed a ects ia een agenesis 36 Define the plate a 36 Statie autosampler acco oe Secs opal ea saab edna inte cohen ewahoaence 38 Load the plate 220 00 ec ccc cece ec cceceeceeceectececetectececeeeceeseeeee 39 PUNO SR ese ae ore tgs et coe eat Saad SAR iti aeDe eee een ta dead 39 REDOR eae ne Ce een aa RE 40 Table of Contents Chapter 1 General Information and Safety Chapter 1 General Information and Safety
59. x Load Matrix View Matrox Edit Matrix Clear Matrix Create Matrix Starts the compensation wizard Load Matrix Apply an existing compensation matrix View matrix Opens the compensation matrix view Edit matrix Opens the compensation matrix for editing Clear matrix Stops applying a compensation matrix Advanced menu For field service personnel only Help Access the current software version number About Software information including version number amnis part of EMO Millipore INSPIRE for the FlowSight Help Opens the user manul in a web browser 43 Chapter 4 Troubleshooting Chapter 4 Troubleshooting This section is designed to help you troubleshoot the operation of the FlowSight If additional assistance is required contact the Amnis service department System Unstable fluidics Air or clog in system Fluidics respond sluggishly Event rate slows over time Event rate is slower than expected Cross contamination from previous samples Erroneous fluid level indicator Instrument will not pass ASSIST Compensation fails to compute values and save files Software INSPIRE appears to freeze INSPIRE Fails to launch Plots fail to update or update slowly Data file fails to collect Image No images Imaging and acquisition rate is erratic or appears frozen Objects appear streaked Objects are not centered in the channel Objects are rotating in the core stream O
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