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Attune® Acoustic Focusing Cytometer User Guide

1. xan ED azm Compensation Matrix Compensation Matrix You can view manually edit or reset compensation matrix values here xan azam Attune Acoustic Focusing Cytometer User Guide OQ IMPORTANT Once you have recorded all compensation controls and calculated and applied the compensation matrix you cannot adjust the PMT voltages for experimental data Note The Compensation Matrix is the inverse of the Spillover matrix To view the Spillover Matrix open the FCS viewer and select the Spillover tab Note If you need to adjust voltages after recording any of the compensation controls you need to remove the data from all compensation controls containing recorded data by right clicking on the control sample and selecting Clear Control Sample Data or right clicking on Compensation in the Experiment Explorer and selecting Remove Compensation If you opt for the latter you have to re run Compensation Setup to recreate all compensation control samples Attune Acoustic Focusing Cytometer User Guide 29 Generating 1 To generate a Compensation Matrix without control samples click the Use Compensation Compensation button Matrix without 2 The following dialog box opens Control Samples u Attune Cytometric Software This experiment doesn t have a compensation setting
2. 6 Close the syringe pump door N Note Proper syringe to valve seal is crucial for the operation of the cytometer when fluids are cycling through the system Cavitations may occur if a seal is not properly attained Attune Acoustic Focusing Cytometer User Guide 49 Appendix A Ordering Information The reagents and consumables supplied with the Attune Acoustic Focusing Cytometer are also available separately from Life Technologies Ordering information is provided below For more information go to www lifetechnologies com or contact Technical Support N Note Refer to the Applied Biosystems Attune Auto Sampler User Guide Part no 4479066 for information about reagents and consumables for the optional Attune Auto Sampler Product Amount Cat no Attune Focusing Fluid 1X Solution 1x1L 4449790 6 x 1 L 4449791 Attune Focusing Fluid 10X Solution 1x1L 4449792 Attune Wash Solution 500 mL 4449755 Attune 10X Shutdown Solution 250 mL 4454955 Attune Performance Tracking Beads 50 measurements 4449754 50 Attune Acoustic Focusing Cytometer User Guide Attune Acoustic Focusing Cytometer User Guide 51 Appendix B Performance Tracking 0Z Performance Tracking Baseline Calculations Performance Test Reports Note Results of instrument performance tracking tests are available for all users but only users authorized by the administrator can per
3. Attune Acoustic Focusing Cytometer User Guide Jo e You can also use the Compensation Slider Bars to assist in fine tuning the Compensation To use the compensation slider bars compensation must be turned on The slider bars are available during sample acquisition and recording so the sample can be tweaked in real time Lysed Whoke Hind e Hovering over the slider bar handle will show the compensation value e To use the compensation sliders drag the appropriate slider bar to adjust the population of interest Only a subset of events is displayed while the slider bars are used to provide real time movement e Adjusting the slider will move populations in the direction the slider bar is moved on that axis e There are two adjustment modes o Course Adjustment will move the slider bars in 1 unit increments up 25 units in each direction To use course adjustment just drag the slider in either direction o Fine Adjustment will move the slider bars in 0 1 unit increments up to 2 5 units in each direction To use the fine adjustment use Shift drag or the arrow keys e Upon release of the mouse all plots will refresh ys Note Adjusting the values in the matrix may adversely affect the compensation in other channels Attune Acoustic Focusing Cytometer User Guide Collect Data After you have calculated the compensation settings you are ready to run your samples to acquire and recor
4. Sample probe is not centered in the sample tube SIP tube is bent Carefully bend the SIP tube in place SIP tube is faulty Contact Technical Support Sample tube is not aligned vertically on tube lifter Readjust the sample tube on the tube lifter Focusing fluid pump does not shut off Focusing fluid filter is clogged Replace the focusing fluid filter Focusing fluid reservoir level sensor is malfunctioning Perform Stop function If the problem persists shut off the instrument and contact Technical Support Fluid is leaking from the base of the instrument or into the drip tray Crack in fluidics tank Replace the damaged fluidics tank Snap fitting is broken or dripping Contact Technical Support Fluidics valve failure Turn off the instrument and contact Technical Support 10 mL or 1 mL syringe seal is broken Contact Technical Support Focusing fluid filter is leaking Replace the filter Attune Acoustic Focusing Cytometer User Guide 143 Performance Tracking Troubleshooting Observation Possible Causes Recommended Solutions High APMT ina single channel Improper bandpass filter in channel Check the optical configuration Improper dichroic mirror placement Check the optical configuration Scratched or defective bandpass filter or dichroic mirror Clean filters if problem persists contact Technical Suppo
5. Parameters Threshold Voltage Custom Parameters Advanced Options Parameters Threshold Voltage r Custom Parameters Advanced Options ___ Please enter a voltage for each parameter mv FSC ssc BL1 BL2 Please enter a voltage for each parameter mV BL3 VvL VL2 FSC SSC BLi BL2 1600 2100 2300 2200 2550 1250 _ 1950 BL3 BL4 RLI 1600 p100 2300 2200 2550 2550 1250 The range of voltage settings is 0 4 999 mV for all channels e To set the threshold and voltage values adjust the slider bars below each detector or enter the desired number directly in the appropriate window 110 Attune Acoustic Focusing Cytometer User Guide Custom Parameters Tab Advanced Options Tab Custom Parameters tab allows you to create derived parameters Instrument Settings Experiment 06 16 2010 02 36 PM Instrument Settings You can use the following options to update instrument settings Parameters Threshold Voltage Custom Parameters Advanced Options Name Oper 1 Param 2 e You must define a derived parameter prior to acquisition A custom parameter cannot be created for previously recorded data S a e Only parameters that are enabled see Parameters Tab page 109 are available in the drop down parameter list lt as Advanced Options tab allows users with administrative pr
6. o 617 o 10 o 10 10 o Lian _or ten ten ten Ie ten Is J Threshold settings Sets the minimum signal level for each detector to eliminate unwanted events and reduce noise The software records and analyzes only the events with parameter values above the set threshold You can also combine all thresholds using Boolean operators e Ifthe And operator is selected all the thresholds set will have to be met before the data is collected e Ifthe Or operator is selected data collection begins when at least one of the thresholds is met e If Ign ignore operator is selected no threshold is set for that detector Note To record events using pulse width you must set a gt 0 threshold value in the respective channel s Voltage mv FSC SSC BLi BL2 BLS VLI VL2 VL3 160 210 230 220 255 125 195 22o Voltage settings Selects the voltage for each detector The range of voltage settings is 0 4 999 mV for all channels To set the threshold and voltage values adjust the slider bars below each detector or enter the desired number directly in the appropriate window Note You may also adjust the threshold and voltage settings by selecting Instrument Settings window on the View Tab 100 Attune Acoustic Focusing Cytometer User Guide Experiment Experiment Explorer lists Experiments in a hierarchical view and
7. 041310 AM 1000 B VL1 H Hi NEV 02 10 10 VLI H Count 800 600 8 400 i Count ga 041310 AM 041310 AM FSC A 10 6 SSC A 10 6 1000 Ei BIZA N NEV 102 BL3 A B AM La 10 10 10 BL2 A Count 041310 AM 041310 AM 1000 VL2 A VL3 A 400 8 200 W 10 10 10 VL2 A VL3 A 041310 AM 041310 AM 40 SC H z 30 50 8 20 i 10 0 00 05 10 15 20 SSC H 10 6 041310 AM 1000 EE 0 ans fi A al ie 102 40 10 102 10 10 BL2 H BL3 H 00 05 10 15 20 FSC H 10 6 041310 AM Count 041310 AM 041310 AM VL2 H 600 VL3 H 5 400 S 200 o 02 04 10 VL2 H VL3 H 127 Local Statistics Local statistics are plot specific and display gating hierarchy specific to the selected plot In the example below the gating hierarchy specific to the BL1 A plot parent and children data is displayed Parameters BL1 A vs Count Gate Singlets A Experiment Name 4 Peak Beads Specimen Name Specimen Sample Name 20100420 Name Event Count BL1 A Mean BL1 A C V E Dai Events 12 012 1 042 578 196 13 L Singlets A 10 281 687 482 142 29 Ee L BL1 A 2 552 2 047 367 2 00 El FSC A 2 552 2 047 367 2 00 O SSC A 2 552 2 047 367 2 00 128 a EC A E Tr 5 Ti 1 mm o0 05 i 15 oo 05 16 15 2 PoC A LI k FILA LOG Attune Acoustic Focusing Cytometer User Guide Displaying Displaying
8. Contact Technical Support Fluidics system is dirty Run Wash function Improper filter placement Check the optical configuration Laser is misaligned Contact Technical Support Laser delay calculated incorrectly Contact Technical Support Bubbles in the system Run De bubble function Improper dichroic mirror placement Check the optical configuration Emission filters swapped Check the optical configuration Attune Acoustic Focusing Cytometer User Guide Sample Troubleshooting Observation Possible Causes Recommended Solutions Weak or no fluorescence from the sample Insufficient antibody present in sample Ensure adequate antibody concentration for the total number of cells stained by titration Target may not be accessible to the antibody i e intracellular target Ensure that the fixation and permeabilization conditions are optimized for the target Inaccurate choice of fluorochrome Use bright fluorochromes for dim markers Too much spectral overlap Use fluorochromes that are spatially separated Incorrect compensation Ensure that the positive single color control is set up correctly on the flow cytometer and gated compensated correctly to capture all the events Target not present or expressed poorly Ensure that the sample expresses the target protein and allows its detection Experiment is not optimized
9. System Specifications Physical Characteristics Optics Fluidics Electronics Computer User Interaction with Instrument Attune Cytometric Software Footprint H x W x D Approximately 16 40 cm x 23 58 cm x 177 43 cm Weight Approximately 64 lb 29 kg Operating temperature 15 30 C Operating humidity lt 80 non condensing Electrical requirements 100 240V AC 50 60 Hz lt 300 W Excitation lasers Blue Violet configuration uses a 488nm 20mW laser and a 405nm 50mW laser and the Blue Red configuration uses a 488nm 20mW laser and a 638nm 50mW laser Alignment Fixed alignment no customer maintenance required Sample input rates 1 sample minute Sample rates 25 1 000 pL minute Cuvette flow velocity 0 5 m second or 2 0 m second Sample delivery Calibrated delivery volumes for volumetric analysis Sample analysis volume 50 uL 4 mL Minimum required sample volume 120 uL Fluid storage Within instrument with level sensing Nominal fluid consumption 1 L day Sample tubes Accommodates from 17 x 100 mm to 8 5 x 45 mm tubes Particle size range 1 0 m to 45 pm Note The minimum sample analysis volume is 50 uL However the system requires that at least 220 uL is used to account for the analysis volume boost volume safety volume Data acquisition Up to 20 000 events second Resolution At least 6 decades PMT voltage User adjustable e Minitower running Windows XP SP3 e 24 inch f
10. e Ensure that the 1 mL sample syringe is sealed properly e Faulty pinch contact Technical Support Run button is not available 140 Focusing fluid tank is empty e Fill the focusing fluid tank e Ensure that the fill lines and fluid level detectors are plugged in completely Lid interlock is not triggered Ensure that the lid is properly closed Tube lifter is not engaged Push tube lifter to up position Instrument is powered off Turn on the instrument Startup not completed Perform Startup USB cable not connected Ensure that the USB cable is plugged in to the instrument and the computer Sample tube is not selected Select the sample tube Attune Acoustic Focusing Cytometer User Guide Observation Possible Causes Recommended Solutions Computer is not communicating with the instrument USB cable not fully plugged in Examine the USB plug in the back of the instrument and the computer Faulty USB cable Contact Technical Support USB port changed from the original port Try different USB ports until communication is restored If the problem persists reinstall the USB drivers Instrument and or computer has no power Power supply not plugged into the appropriate outlet Ensure that the instrument and or computer are plugged into the appropriate outlet No power at the outlet Make sure that the outlet is functio
11. oo pono pn nan nnn n t j i u ee Attune Acoustic Focusing Cytometer User Guide 43 Plots The Attune Cytometric Software displays data in histogram dot or density plots Histogram istoga Histogram is a graphical representation of single parameter data and shows the relative number and distribution of events In a histogram the horizontal axis corresponds to the signal intensity of the selected parameter while the vertical axis represents the number of events count Count Dot Plot Dot Piot A dot plot is a graphical representation of two parameter data where each axis represents the signal intensity of one parameter Each dot in the plot corresponds to one or more events detected above the threshold SSC A 10 6 jw cn FSC A 10 6 Density Plot Density Plot a A density plot is a graphical representation of two U parameter data where the colors represent the collection of events with the same intensity and each axis represents the signal intensity of one parameter SSC A 10 6 jw cn FSC A 10 6 See Customizing Plots on page 115 for more information 44 Attune Acoustic Focusing Cytometer User Guide 3 System Maintenance Maintenance Schedule The Attune Acoustic Focusing Cytometer is designed to require minimum maintenance However to ensure reliability of the cytometer you must perform basic preventative maintenance procedures on a reg
12. 132 Attune Acoustic Focusing Cytometer User Guide Software Administration Several functions of the Attune Cytometric Software are available only to the software administrator or to users designated by the administrator Administrator Options while General Options are user specific and available to every user Administrator Administrator options are only accessible to the software administrator or Options designated users 1 Click Options Lath shortcut button in the Quick Access toolbar Alternatively click Options under Global in the Home tab ag zs e Optons Main Transaction Menu Log Global Options window opens Options You can use this to manage users and general application settings General amp Advanced options for working with Attune Cytometric Software User Management Folder Locations 2 _aH AA _i T 5 5 5 86Ta T T TTTZLTZEUU gt D U_UU_U U___77 jU___ Styles Users C Program Files Applied Biosystems Attune Users Resources Shared Templates C Program Files Applied Biosystems Attune Shared Templates Performance Tracking C Program Files Applied Biosystems Attune Performance Tracking Reports C Program Files Applied Biosystems Attune Reports Other Settings Institution Name Instrument Name Instrument Serial Number 2 Click Administrator to assign the following folders e Users e Shared Templ
13. Workspace Workspace displays Analysis objects plots gates and statistics e Workspace types o Global L Workspace is common to all samples in an experiment o Local Workspace is unique to a sample and is denoted by an asterisk on the sample o Compensation Workspace defines compensation controls and is pre defined e Creating a New Experiment from the explorer uses the default Workspace as the Global Workspace unless you specify another Workspace e Creating an experiment using a template uses the Workspace saved with the template e Double clicking any Sample lil or I in the Experiment Explorer opens the Workspace Global or Local associated with that Sample e Double clicking the Lil icon opens the Global Workspace for that experiment e The Global Workspace can be modified by directly opening the Global Workspace Lull or at the Sample lll level To apply sample level changes to the Global Workspace right click on the Sample and select Save Workspace as Global e The Compensation Workspace is accessed via the Compensation Setup Guide or by double clicking on Compensation Ml New screen RB nt d Attune Cytometric Software v1 2 5 Admin se qe a il Wla E Stop recording when Any of these conditions are met 10000 Events on All Gates Sample 1 11 15 2010 01 10 PM gt 11 16 2010 11 09 AM 1 0 ev sec 0 events p gt 11 16 2010 11 18 AM Violet Laser Sample
14. er s Note Options menu allows authorized users to perform administrator and user management functions determine file and folder pathways and change the default settings for general formatting Attune Acoustic Focusing Cytometer User Guide Insert The Insert tab enables you to insert analysis objects i e plots and gates as well as text images and statistics into the Workspace Insert Compensation View Plate w A Te Load Events ral E j Da All Events v Histogram Dot Density Rectangular Oval Polygo Quadrant Derived Histogre Bi Marke Text Image Statistics Plot Plot Plot sate sate sate Gate sate Sate Plots Gating Tools Other Analysis The Insert tab consists of Plots Gating Tools and Other e Plots allow you to insert a Histogram Plot Dot Plot or a Density Plot into the Workspace e Gating Tools allow you to isolate a region in a selected plot for analysis e Other allows you to display the statistics corresponding to a selected plot in the Workspace It also allows the insertion of text and images into the Workspace To display global statistics click Statistics without selecting a plot Global statistics contains data of all the gates in the Workspace To display local statistics select a plot in the Workspace and then click Statistics Local statistics only displays data pertaining to the selected plot Compensation The Compensation tab allows you to access the compensation functions of th
15. AAA AA 2 k k Z _ Enable bubble sensor Laser Delay Sets the time delay between the two lasers which is calculated during the Performance Test Area Scaling Factor Blue and Violet or Red and Blue Sets the value to scale height and area to equivalent values Area scaling is calculated during the Performance Test Enable bubble sensor The bubble sensor is enabled by default but an administrative user may wish to disable the sensor N Note Save As saves the Instrument Settings as a printable PDF file The file contains all the parameters voltage threshold settings and hardware codes The report does not contain the instrument name institution or compensation matrix if created Attune Acoustic Focusing Cytometer User Guide 111 Customizing the Attune Desktop The Attune Cytometric Software allows you to design the layout of the Attune Desktop and how the different components are positioned The Attune Desktop is designed to be customizable to your needs and works similar to other Windows based applications You can choose to show or hide the different components by checking the appropriate boxes in the View tab see page 106 The Attune Desktop is configured per user Moving and e You can move each panel or component of the Attune Desktop as desired Resizing e To delete an item such as a chart from the Workspace simply click to select it Workspace Objects and then
16. Access Type Operator _ Enable Viewing System Transactions Enable Viewing User Transactions Enable Running Baseline Calculations Enable running Performance Test 134 Attune Acoustic Focusing Cytometer User Guide Removing Users Changing and Resetting Passwords Changing User Privileges Changing Default Styles Enter appropriate information and permissions Assign password by selecting Change Password Enter new password and confirm Click Options itt shortcut button in the Quick Access toolbar Alternatively click Options under Global in the Home contextual tab Only a user with Administrator privileges can remove users Click User Management Select the user you want to remove and click the Trash icon W Click Options Lat shortcut button in the Quick Access toolbar Alternatively click Options under Global in the Home contextual tab Click User Management Select the user from the list displayed Click Change Password to assign a new password or click Reset Password to reset an existing password Enter new password and confirm Note If a password was not assigned to a new account the user will be prompted to create a password when logging in for the first time OQ IMPORTANT User accounts are stored in an encrypted file Lost and forgotten passwords cannot be recovered An administrator must reset the password if a user forgets their password Click Options La shortcut bu
17. Indicates a terminal that can receive or supply alternating or direct current or voltage 150 Attune Acoustic Focusing Cytometer User Guide Safety Symbols Environmental Symbols on Instruments The following table describes the safety symbols that may be displayed on Applied Biosystems instruments Each symbol may appear by itself or in combination with text that explains the relevant hazard see Safety Labels on Instruments on page 152 These safety symbols may also appear next to DANGERS WARNINGS and CAUTIONS that occur in the text of this and other product support documents Symbol Description Indicates that you should consult the manual for further information and to proceed with appropriate caution Indicates the presence of an electrical shock hazard and to proceed with appropriate caution Indicates the presence of a hot surface or other high temperature hazard and to proceed with appropriate caution Indicates the presence of a laser inside the instrument and to proceed with appropriate caution Indicates the presence of moving parts and to proceed with appropriate caution Indicates the presence of a biological hazard and to proceed with appropriate caution Indicates the presence of an ultraviolet light and to proceed with appropriate caution gt P gt The following symbol applies to all Applied Biosystems electrical and electronic products placed on the European market after A
18. Peug Pe 29 530 30 574 26 Forward scatter irs 405 10 640LP 5 e Fesc 522 31 Side scatter 450 40 v VL2 Pvug Violet 405nm Laser N IACUE E Eg 20mw E7SSCI ERT EBL3T Lem Red 636nm Laser 50 m vv Blue 488nm Laser 20 mv Notes 1 At Side Scatter location the neutral density ND filter is in a user removable filter holder and the 405 10 filter is in a non user removable filter holder tube Attune Acoustic Focusing Cytometer User Guide 85 Instrument Exterior Components Power Status Indicator Switch Lights AB Attune E Sample Injection Port Fluidics Compartment Sample Injection Port SIP Sample Injection Tube Sample Tube Lifter 86 Attune Acoustic Focusing Cytometer User Guide Instrument Interior Components Fluidics The images below show the fluidics compartment of the Attune Acoustic Focusing Compartment Cytometer Fluid Lines Fill Sensor Connections Focusing Fluid Shutdown Fluid Compartment Compartment l Waste Focusing Wash Shutdown Waste Container Wash Fluid Fluid Fluid Fluid Compartment Compartment Syringe Pump The image below shows the syringe pump compartment of the Attune Acoustic Compartment Focusing Cytometer Syringe pumps Focusing fluid filter Attune Acoustic Focusing Cytometer User Guide 87 Optics The image below shows the optics compartment of the Attune Acoustic Focu
19. Run Unclog function Defective pinch valve Contact Technical Support Scatter pattern is unclear Instrument settings are not optimized e Optimize experiment parameters for cell type e Ensure that the axis have the same scale Cells were fixed Some reagents used for fixing and permeabilizing the cells alter scatter patterns Prepare new sample using different reagents for fixing and permeabilizing the cells Bubbles in the fluidics system Run De bubble function 142 Attune Acoustic Focusing Cytometer User Guide Observation Possible Causes Recommended Solutions High CV with sample Poor sample preparation e Repeat the sample staining procedure e Optimize reagent stain conditions Flow cell may be dirty Perform system wash or Shutdown cycle Air bubble in flow cell Run De bubble function Incorrect laser delay blue channels only Clean the cytometer optics and re run Performance Test Sample aspirated then backfilled into sample tube Clog in the sample line Run Unclog function Pinch valve error Contact Technical Support Pump error Contact Technical Support Long delay between sample aspiration and events appearing on screen normally events appear in 10 seconds Sample syringe is leaking Ensure that the sample syringe is sealed properly Partial clog in the fluidics system Run Unclog function
20. S C k 8 ST _ Compensate on Area O Height aArea Height For these control samples _ ___ For these control samples __ Unstained Control Unstained Control Mv BL1 BL1 BL4 z v L2 BL2 BL2 RL1 Mv BL3 BL3 RL2 26 Attune Acoustic Focusing Cytometer User Guide 2 Select the type of compensation you want to perform and the desired channels and click OK Compensation Setup Guide opens and the Workspace is automatically populated with the plots necessary to calculate compensation Thos Com peo buy Load Unetained Control Gortra Click Run Scmther Click Record Action Gate Stop eccording mia Posttion Gate Ary of these con 10000 Evan 415 Mn D an rest ahan Hha d zq 254 4000 pl 4 N Racord cor rarc nb E a i qd 3 i t Karar One Creel at lamt SOU T eens wf Rengat arisi ne ROM OES R n Record Stop Oar VL1 A Hew ampe gt i Sample Duperaad axak k n Volume JM LL EBI LL botal draw vieme 40 ar sitii 100 pimin i i W 4 LI A ak EF E jarn ys Note You can move the Compensation Setup Guide anywhere on the Workspace by clicking on its title and dragging it across the Workspace Y Note You can remove compensation channels by right clicking on the compensation control under Compensation in the Experiment Explorer and selecting Delete Control Sample You can a
21. To ensure that only the desired criteria are used deselect the unwanted criteria The collection controls allow you to prompt the Attune Acoustic Focusing Cytometer to run your samples and record flow cytometric data using seven basic commands Run Record Pause Stop Clear Save and Next Sample e Run Commences data acquisition without ea recording You can observe the events in real b ek ae time as they are plotted in the Workspace Run Record a Clear allowing you to adjust experiment settings such as the threshold parameters and PMT voltages e Record Initiates the recording of data in 7 FEA SS FSR a file T e 7 e Pause Pauses data acquisition or recording ee ee oe ee It also pauses the pumps If you click Pause during Run mode the Save icon will appear e Stop Terminates data acquisition or recording If you click Stop during Run mode the Save icon will appear Clicking Stop during Record mode will stop the run Sane 45 Save CEGAH Stop and save the data up to that point of the acquisition Next NextSample gt gt e Clear Deletes the data from the screen It refreshes the Workspace while the instrument is in the Run or the Record mode e Resume Resumes a paused run e Save Saves the last 20 000 collected events to the FCS file If the sample has existing data you can append or overwrite the existing data e Next Sample Moves to the
22. a g E K K o g K g a g E K K K K E K K K K K K K e The software records and analyzes only the events with selected parameters default is all but Include Event Count selected e To select or deselect parameters check or uncheck the desired parameters e Renaming parameters from the Parameters tab changes the parameter name within the FCS file OQ IMPORTANT If you are acquiring greater than 500 000 events you must limit the number of acquisition parameters to 10 or fewer Note that Event Count and Time are also considered parameters For example you may select Area for all channels plus Event Count and Time for a total of 10 parameters but not Area and Height for all channels which would total 16 parameters Attune Acoustic Focusing Cytometer User Guide 109 Threshold Tab Threshold tab allows you to set the minimum signal level for each detector to eliminate unwanted events and reduce noise The software records and analyzes only the events with parameter values above the set threshold Instrument Settings Experiment 06 16 2010 02 36 PM Instrument Settings Experiment 07 01 2011 09 34 PM instrument Settings Instrument Settings You can use the following options to update instrument settings You can use the following options to update instrument settings Parameters Threshold Voltage Custom Parameters Advanced Options Par
23. and then click Search 3 Find the document of interest right click the document title then select any of the following e Open To view the document e Print Target To print the document e Save Target As To download a PDF version of the document to a destination that you choose Q IMPORTANT For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer 160 Attune Acoustic Focusing Cytometer User Guide Attune Acoustic Focusing Cytometer User Guide 161 EDA MIN a Headquarters d 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit www appliedbiosystems com support technologies www lifetechnologies com
24. dilute sample In a conventional cytometer the diameter of the sample core is varied by the pressure difference between the sample stream and the sheath fluid stream Increasing the sample flow rate enlarges the core diameter which allows faster data acquisition but lower resolution because the cells can float freely in the sample core stream and may pass through the laser spot off center In contrast the alignment of cells in the Attune Acoustic Focusing Cytometer is independent of the total fluid flow through the cytometer While large changes in the amount of sample injected or the total fluid flow may alter the diameter of the sample core this does not affect the resolution of the cytometer because constant focusing ensures that the cells remain in the optimal position for interrogation by the aligned laser This feature of the cytometer allows even dilute samples to be analyzed at a high rate without a loss of resolution The Attune Acoustic Focusing Cytometer has a maximum flow rate of 1 mL minute but we recommend that you run your samples at a collection rate of 25 500 L minute for the best results Attune Acoustic Focusing Cytometer User Guide 73 If high sensitivity is more important than throughput the flow rate can be decreased four fold by switching the cytometer to high sensitivity mode to maximize the collection of fluorescence photons which are dependent upon the excitation intensity and cell transit time Samp
25. 4 11 16 2010 11 18 AM 1 Workspace B Instrument Settings 4 ny Compensation Scatter Sample Blue Laser Sample 0 ee Ea unstained Control gt 4 ad 1 nsan R an Ms Min o Sec Ro a a a aad Hi Z 4ooo e j s 2 hl O Record continuously lt ji B13 Ri p a ewaya den 4 MESES a v 7 4 E va 0 10 o 0 10 10 10 10 10 ji v 2 gt m ii Ii v Run Record Stop 4 dk Specimen Parameters FSC H vs SSC H mag Nasan gt Gate Ungated Ti Sample e mple n P Experiment Name New Experiment IC 11 18 2010 09 22 AM 5 Speamen Name Specimen VERE US denli Disa Sk 11 18 2010 03 20 PM Sample Dispensed OyL Name Event Count Parent Total 4 11 11 2010 12 34 PM 1 Jal Events 40 405 100 000 Acquisition Volume 300 yL 470 n hal Workspace Or 39 980 98 48 98 948 2 F Instrument Settings andar io ligh Sensitivity AES ELLS i Compensation 100 uL min a 11 11 2010 12 34 PM 2 Workspace g Collection Panel Instrument Confi B Instrument Settings m fe va BN I AA Workspace Default Instrument Settings Default O Status None Compensation None Workspace Attune Acoustic Focusing Cytometer User Guide 103 Ribbon OB Home Insert Clipboard 104 The Attune Cytometric Software uses the Microsoft Ribbon interface The Ribbon consolidates related functionality
26. AND IS NOT TRANSFERABLE THE FOREGOING LIMITATIONS OR EXCLUSIONS OF WARRANTIES LIABILITY REMEDIES OR DAMAGES SET FORTH ABOVE SHALL NOT APPLY TO THE EXTENT PROHIBITED BY LAW Attune Acoustic Focusing Cytometer User Guide 147 Appendix G Safety This section includes the following topics e Safety conventions used in this document e Symbols on instruments e Safety labels on instruments e General instrument safety e Chemical safety e Chemical waste safety e Electrical safety e Physical hazard safety e Biological hazard safety e Laser safety e Workstation safety e Safety and electromagnetic compatibility EMC standards e SDSs 148 Attune Acoustic Focusing Cytometer User Guide Safety Conventions Used in this Document Safety Alert Words Four safety alert words appear in Life Technologies user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action Definitions IMPORTANT Provides information that is necessary for proper instrument operation accurate installation or safe use of a chemical CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injur
27. Do you wish to create a default one or open the compensation setup window Create Default Compensation Setup 3 Select Create Default to create a blank matrix The matrix can now be edited by opening the Compensation Matrix and entering values in the appropriate fields of the table 5 Alternatively you can use the Compensation Slider Bars to adjust the matrix See Applying Compensation on page 33 30 Attune Acoustic Focusing Cytometer User Guide Generating Compensation Matrix Using Previously Recorded Data To generate a Compensation Matrix using previously recorded compensation controls select Compensation from the main menu then select Compensation Setup Guide and select Import FCS file in the Compensation Setup Guide window Compensation Setup Guide Import FCS file Show Compensation Setup Load Unstained Control Click Run Click Record Position Gate L Do not show this dialog again Close Alternatively right click the appropriate compensation control sample and select Import FCS file ji WLI Delete Control Sample u EKEXIi XEWE Set the scatter gate on the desired population and the corresponding histogram gate on the positive peak If no samples have been recorded the experiment Instrument Settings will automatically be updated and grayed out to prevent any changes to the voltages If samples have already been recorded and have voltages different from
28. Local Statistics Statistics Select a plot and click on the Statistics icon in the Insert tab of the Ribbon menu to insert a Local statistics box for that plot Alternatively right click on a plot and select Statistics Scatter test sample 250 Cut Ctri X 200 Copy Ctrl C Delete Del 150 5 Duplicate O Save As 100 50 Statistics 0 02 0 0 4 New Gate b 1 1 1 Set Population gt BL2 A Customize Displaying Global Statistics Click on the Workspace and then click on the Statistics icon in the Insert tab of the Ribbon menu to insert a Global statistics box Alternatively right click on the Workspace and choose Insert gt Statistics to insert a global statistics box Select All Chrl A Globally Use Compensation Reset Compensation Matrix Histogram Plot Dot Plot Density Plot Text Image Attune Acoustic Focusing Cytometer User Guide 129 Customizing To customize statistics boxes right click the statistics box and then select Statistics Customize a A number of standard statistics You can use the following options to customize workspace items me asurements are availab le i e Event Count Header Tunga WI s s Show ZE TU TT T Experiment specimen Median B r u Av 3 v E Sample Ome Recorded e Mean Population Tahoma lw gi Show _ vent Coun vent Percent Paren Far haere eweyan e Event Percent Parent Mean C Standard Deviation E e Event P
29. Multiple Copy and Paste Functions Customizing Text Objects Customizing the Compensation Workspace 114 You can paste multiple copies of an object or different objects onto the work area When pasting objects the most recently pasted item will be on the top Violet Laser Sample Violet Laser Sample 2 H 2 H Right click on the text object and select Customize to open the Customize dialog box The Compensation Workspace is pre populated with plots corresponding to the compensation controls selected during the compensation setup e The Layout of the Compensation Workspace cannot be changed e Objects that can be customized include the R1 and R2 gates and the scale and range of all plots e To customize the scale of the X and Y axis right click on the axis and choose Scale gt Linear or Scale gt Log e To customize the scale and range of the X and Y axis right click anywhere on the plot and choose Customize Customize You can use the following options to customize workspace items X Axis ______ Y Axis Parameter FSC A Parameter SSC A Scale Scale Type tines i wll Type Linear E Range Automatic Range Automatic Manual Manual Minimum o Minimum o e The Linear scale is the only option on the Y Axis of the histogram plots Attune Acoustic Focusing Cytometer User Guide Customizing Plots Plot Context Context menus can be used to open the plot m
30. Six PMTs are dedicated to fluorescence detection one PMT detects signals from SSC and FSC is obtained from a diode detector The digital electronics of the cytometer amplify and analyze these pulses and transfer them to the workstation computer for further processing by the Attune Cytometric Software Results Results are saved as FCS 3 0 files in the appropriate user folders Attune Acoustic Focusing Cytometer User Guide 71 Fluidics Schematics of the Fluidics System iz The fluidics system of the Attune Acoustic Focusing Cytometer establishes the fluid flows required for acoustic focusing cytometer operation This includes the flows during the main data collection operation as well as during the Startup Wash Unclog Rinse Run De bubble Stop and Shutdown operations The sample to be analyzed is driven by a syringe pump and passes through the bubble sensor and the sample loop before arriving at the capillary assembly A separate pump forces the focusing fluid through the focusing fluid filter and flow dampener The capillary assembly is an acoustic resonant device that focuses cells or particles in the sample fluid into a single tight line i e the sample core using a capillary coupled to a single piezoelectric transducer The capillary carries the sample core upward through the center of the optical cell where the particles to be analyzed are intercepted by a tightly focused laser beam for interrogation After passin
31. a L ji Instrument is not detected USB cable not connected or the cytometer is not Powered on Tube lifter is not engaged in the top position Note The Instrument Status Bar displays a status indicator icon only if the cytometer encounters the specific error symbolized by the icon K reha E gt Ve d LF Functions menu allows you to perform e De bubble e Rinse e Clear Error e Wash e Startup e Status e Unclog e Stop Zoom tool allows you to zoom in and out of the Workspace and to scale it to fit a single page for printing when Print Area is visible Attune Acoustic Focusing Cytometer User Guide 107 Quick Access Toolbar AB tab 108 Quick Access Toolbar contains the Undo Redo and Options buttons Home Insert Undo deletes the last action Redo restores the last action Options opens the Options window which is used to customize the Attune Cytometric Software Some options are user specific while others are application specific i e global to all users and customizable only by an authorized user Show Main Menu Screen opens the Main Menu Screen Customize Quick Access Toolbar allows you to change the location of the Quick Access Toolbar or to minimize it Note You can add any button in the Ribbon and within the AB Button to the Quick Access Bar by right clicking the button and selecting Add to Quick Access Bar HP The AB tab al
32. assembly is an acoustic resonant device that focuses cells or particles into a single tight line using a capillary coupled to a single piezoelectric transducer Acoustic focusing exploits the size density and compressibility differences between cells or particles relative to the background carrier medium to position the particles or cells into a single focused line along the central axis of a flow channel Because acoustic focusing is independent of sample fluid flow the Attune Acoustic Focusing Cytometer allows a tight control over the sample flow rate which in turn permits the control of interrogation time enabling higher sensitivity and precision As the sample traverses the interrogation point the Attune Acoustic Focusing Cytometer uses a violet and a blue laser or a red and a blue laser to illuminate the particles or cells in the sample which scatter the laser light and emit fluorescent light from fluorescent dyes attached to them The optical filters and mirrors route specified wavelengths of the resulting light scatter and fluorescence signals to the designated optical detectors Attune Acoustic Focusing Cytometer User Guide Signal Processing The Attune Acoustic Focusing Cytometer has seven PMT detectors and one Diode detector FSC for converting the fluorescence signals and collected light scatter into electrical signals i e voltage pulses which are proportional to the intensity of the light received by the detectors
33. button If the matrix was created manually clicking Reset reverts the matrix to the null no compensation matrix Attune Acoustic Focusing Cytometer User Guide Applying By default compensation is ON when there is a compensation matrix available Compensation e Globally Use Compensation turns compensation ON and OFF for all plots in the Workspace When the button is lit compensation is applied to all plots When it is unlit all plots will show uncompensated data e Use Compensation turns compensation ON and OFF via a parent plot for all descendent plots You must select the parent plot for this button to be active If all fluorescent plots are gated from one parent then this button acts like the Globally Use Compensation button e You can view the Compensation Matrix and manually adjust compensation values by selecting the Compensation Matrix and then manually entering the values for the parameter of interest The Blue Violet configuration shows VL1 VL2 VL3 BL1 BL2 and BL3 The Blue Red configuration shows BL1 BL2 BL3 BL4 RL1 and RL2 Compensation Matrix Compensation Matrix You can view manually edit or reset compensation matrix values here Compensation Matrix Compensation Matrix You can view manually edit or reset compensation matrix values here Area
34. from clogging the fluidics system Perform system flush if you intend to leave the cytometer shutdown for longer than two weeks 1 Replace all fluidic containers focusing fluid wash and shutdown tanks with deionized water 2 Run Startup 3 Run the Shutdown function using deionized water on the SIP instead of bleach 4 Empty all fluidic tanks and allow to dry Attune Acoustic Focusing Cytometer User Guide 2 Running Samples Workflow Before You Begin Sample Requirements Startup Execute Performance Tracking Function Verify Optical Configuration Inter User Wash Procedure Create an Experiment Optimize the Experiment Calculate Compensation Collect Data Attune Acoustic Focusing Cytometer User Guide 15 Before You Begin Sample Requirements Startup Performance Test Verify Optical Configuration Before starting an Experiment you should be familiar with e Attune Acoustic Focusing Cytometer Startup Performance Tracking and Shutdown procedures e Attune Cytometric Software Workspace and Experiment Explorer e You need at least 220 uL of sample in a single cell suspension e The Attune Acoustic Focusing Cytometer is designed to handle samples in tubes ranging from 17 x 100 mm to 8 8 x 45 mm e The method used to prepare a specimen depends on the sample type and the assay desired e In general the maximum recommended sample concentration for analysis is 1 x 10 cells mL If th
35. functions as an Explorer interface for creating new Experiments and recording data e Experiments are groups of specimens Fase ee a ee ea and samples that share common 4 L My permet 2 Workspaces instrument settings and C 12 14 2011 09 27 AM compensation settings lC 12 26 2011 06 34 PM Browse Experiments 12 27 2011 05 53 AM e Each Experiment lists experiment EEE fundamentals such as the global b Global Workspace amp e Wl iiei oe Workspace global instrument settings 4 A Compensation compensation settings specimens and Ai Unstained Control samples Aa BL1 mee e Only one Experiment can be opened at a j4 BL3 time ja v 1 i mo e You can organize your experiments in li vis higher level folders containing multiple a 05 17 2012 02 30 PM experiments Global Works I E Global Workspace e You can rearrange Experiments j Ir KRE Specimens Samples and Folders by a J Specimen drag and drop Hi Sample en Global Instrument Settings My Experiments contains the saved experiments of the user D te Experiment contains the experiment fundamentals of a single experiment which are grouped under the Workspace Instrument Settings Compensation and Specimen icons see below a Global Workspace represents the area that displays Analysis objects plots gates and statistics views Global Instrument Settings allows you to optimize instrument settings such as Selecting P
36. get Voltage Hpcy 9 Violet FSC 700000 72677200 1075 4 80 NIA NA NIA Criteria Met Violet ssc 700000 643589 00 1100 5 80 NIA N A N A Criteria Met Blue BL1 2000000 2031977 00 1075 5 00 0 069 423 262 999 Criteria Met Blue BL2 2000000 200593800 1075 4 80 0 143 76 426 998 Criteria Met f Blue BL3 2000000 2189023 00 1000 360 0 042 136 649 998 Criteria Met Violet vu 2000000 2079497 00 900 600 0 009 267 641 1 000 Borderline i Violet v2 2000000 1890055 00 1075 150 0014 1255 491 984 Criteria Met Violet v3 2000000 185758600 1050 470 0185 5 926 545 Criteria Met RR Functions A LS ls e The panel on the left displays the baseline report history You can access previous baselines reports through this panel Current Baseline 5 17 2012 3 33 PM 756080 Lot No 756080 Expiration Date 05 20 2021 Current Baseline Baseline 5 17 2012 3 33 PM 756080 Previous Baselines Baseline 12 14 2011 9 23 AM 756080 e The Baseline Calculations Report is saved as a PDF file You can use the toolbar above the report to print the report or save it 08 Attune Acoustic Focusing Cytometer User Guide Performance Test After the baseline values are defined the same lot of Attune Performance Tracking Beads is used to run the Performance Tests to measure variation from those baseline measurements to track the daily performance of th
37. more than in the 575 24 filter Attune Acoustic Focusing Cytometer User Guide Electronics Voltage Pulse When a cell or particle passes through a focused laser beam it refracts or scatters light in all directions and can emit fluorescence The scatter and the fluorescence last only a few microseconds because the cells or particles are moving very rapidly through the focused laser beam The detectors convert the momentary flash of light into an electrical signal called a voltage pulse When the cell or particle begins to enter the intercepting laser beam the signal intensity is low because only a small portion of the particle scatters the light The pulse reaches its maximum when the cell or particle is in the middle of the laser beam and the whole particle scatters the light Further the laser beam is brightest in the middle thus causing more light to scatter off of the particle As the cell or particle exits the beam the signal starts decreasing and eventually trails off below the threshold Laser beam Particle or cell O Sample flow Voltage Time Voltage Time Voltage Time tt Voltage Time Attune Acoustic Focusing Cytometer User Guide 81 Pulse The analog signal from the detectors are amplified and relayed to the 24 bit analog to digital converter ADC which samples the signals at a rate of up to 2 5 MHz converting the continuous signal into digital data and producing 6 decad
38. next Sample U Creates a new sample using the global Workspace if no additional samples remain in the experiment Attune Acoustic Focusing Cytometer User Guide The Sample Dispensed options in the collection panel allow you to choose between High Sensitivity and Standard collection modes and to specify the draw volume A status bar displays the sample volume dispensed during a run Sample Dispensed e High Sensitivity Allows sample flow 0 uL rates of 25 uL or 100 uL per minute Acquisition Volume 50 uL 170 uL total draw volume e Standard Allows sample flow rate of ey SE 25 uL 100 uL 200 pL 500 uL and 1 000 uL zaxan per minute Ne Note Using 1 000 uL per minute sample flow rate may require adjustments to instrument settings Attune Acoustic Focusing Cytometer User Guide 99 Instrument Configuration Instrument Configuration allows you to specify Threshold and Voltage parameters for each of the Scatter and Fluorescence detectors on the Attune Acoustic Focusing Cytometer To access click Instrument Configuration tab on the Collection Panel GJ Instrument Configuration Nf Collection Panel Note The example screens below show Scatter and Fluorescence detectors for the Blue Violet configuration For the Red Blue configuration the detectors are FSC SSC BL1 BL2 BL3 BL4 RL1 and RL2 Instrument Configuration ll Xx Threshold x1000 FSC SSC BLI BL2 BLS VLI VL2 VL3
39. to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective eyewear clothing and gloves Read and follow the guidelines in these publications In the U S e U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 www cdc gov OD ohs biosfty bmb14 bmbI 4toc htm e Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx_01 29cfr1910a_01 htm 1 e Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials e Additional information about biohazard guidelines is available at www cdc gov In the EU e Check your local guidelines and legislation on biohazard and biosafety precaution and the best practices published in the World Health Organisation WHO Laboratory Biosafety Manual third edition www who int csr resources publications biosafety WHO_CDS_CSR_LYO_ 2004_11 en Attune Acoustic Focusing Cytometer User Guide 157 Laser Safety Laser The Attune Acoustic Focusing Cytometer blue violet configuration uses a Classification 488nm 20mW laser and a 405nm 50mW laser and the blue red configuration uses a 488nm 20mW laser and a 638nm 50mW laser Under normal operating conditions the Attune Acoustic Focusing Cytometer is
40. when two or more fluorophores are used in an experiment there is often an overlap between the wavelength ranges Compensation is the mathematical method used to correct for the overlap of one fluorophore s emission into another fluorophore s emission measurements Every fluorescent molecule emits light with a particular spectrum unique to that molecule These emission spectra overlap in some cases very significantly The example below shows the emission spectra of Pacific Blue Alexa Fluor 488 R phycoerythrin R PE and Alexa Fluor 647 R PE dyes 488 nm 450 40 v 530 30 575 24 RSR FN Alexa Fluor 488 x R PE x j N Pacific Blue Alexa Fluor 647 R PE I d a Le L EI 440 nm 480 nm 530 nm 560 nm 600 nm 640 nm 680 nm 720 nm Each dye emits with a characteristic emission spectrum that is specific for the fluorophore Alexa Fluor 488 dye has a maximum around 520 nm R PE at about 575 nm Pacific Blue dye at about 454 nm and Alexa Fluor 647 R PE dye at about 666 nm The teal line represents the laser excitation wavelength of an argon ion laser 488 nm In this example the emission spectra of each dye is backlit with a shaded area indicating the emission filter where the specific light is captured on the Attune Acoustic Cytometer In general filters are chosen which collect the emitted light near the emission maximum For example to capture the emission from Alexa Fluor 488 dye a BP 530 30 filt
41. 00 pL minute collection rate run 1 mL deionized water 2 Repeat with 10 bleach solution 3 Repeat with deionized water OQ IMPORTANT 10 Bleach is defined as a 1 in 10 dilution 1 part bleach to 9 parts water of 5 25 sodium hypochlorite in water This gives a final concentration of 0 5 sodium hypochlorite equivalent to 5000 ppm of available chlorine Attune Acoustic Focusing Cytometer User Guide 17 Create an Experiment Using a Blank Template This section explains how to create a tube experiment To create a plate experiment refer to the instructions in the Attune Auto Sampler User Guide Part no 4479066 To run your samples and collect cytometric data you need to create an Experiment in the Workspace The Attune Cytometric Software allows you to e Create an experiment using a blank template e Create an experiment using a pre populated template e Create an experiment from the Experiment Explorer using the default Workspace e Duplicate a saved experiment in the Experiment Explorer You can perform these functions from the Main Menu or by using the Experiment Explorer 1 From the Main Menu screen click Blank Tube Experiment New Experiment No template No template No template No template defined defined defined defined Click here to select one Click here to select one Click here to select one Check here to select one No template No template No template No template No template b defined defined d
42. 40 Compensation 4 12 27 2011 05 53 AM 1 20 20 20 M Global Workspace caspi Sesh 0 0 0 amp Global Instrument Settings 10 10 10 10 10 10 10 10 10 10 10 10 es BL3 A VL1 A VL2 A Nen gt my test 0 lume 4 05 15 2012 11 17 AM Standard VL3 A E Global Workspace Standar 1 a a eY Global Instrument Settings 80 Lan 60 3 40 20 0 VL1 109 02 10 108 VL2 VL3 A VL3 Collection Panel instr ume ent Config ration it m Workspace Default _ e Co S gt Foncions A O tus Non 4 A Specimen Instrument Settings Default mp nsation detaul 11 25 AM 5 15 2012 iy Tel CD You can move minimize or hide the Experiment Explorer the Collection Panel and the Instrument Panel 96 Attune Acoustic Focusing Cytometer User Guide Collection Panel Collection Panel allows you to initiate data collection record events define the collection criteria and select the collection mode and rate The Collection Panel also displays the run status If Collection Panel is not visible select View gt Collection Panel Double click the Sample icon lli to access the Collection Panel Collection Panel ya ar 0 events 4 Run status 0 Stop recording when Any of these conditions are met V 10000 Events on All Gates 7 iv 5 Min 0 Sec W 4000 pL Collection criteria _ Record continuously Other Options Display atleast 10000 v event
43. Compensation Controls Optimizing 1 Double click the Unstained Sample in the a 05 15 2012 11 17 AM Instrument Compensation view in the Experiment Explorer Eg Global Workspace Global Instrument Settings Settings for The Workspace for the Unstained Sample contains ant i ER a ompensation one SSC vs FSC plot containing a polygon gate and histogram plots for each of the fluorescent parameters selected during the compensation setup Unstained Control A Unstained Control Install the unstained control on the tube lifter Sample Dispensed Enter the Acquisition Volume then set the Flow T Rate by adjusting the slider bar e For setup you can conserve your sample by sada running the cytometer in Standard mode at a 25 pL per minute collection rate 4 Click Run Events will appear in the FSC vs SSC plot You can obtain data in real time without saving them to a file Q IMPORTANT DO NOT click Record at this point Attune Acoustic Focusing Cytometer User Guide 23 24 10 Adjust the FSC voltage to place the population on scale by sliding the FSC slider bar up or down Alternatively you can type a specific numerical value in the settings window above each channel Adjust the SSC voltage to place the population on scale by sliding the SSC slider bar up or down Adjust Threshold on instrument control panel to remove unwanted events and background Set the scatter gate on the populat
44. Ge AN 18 PHIM Ze te Ex DeriT l l sy nc a n y a kek VR erkek n Mek kalek Gal k la r N 2A n R 22 Calculate COmpensatl Ons zy e nani n ae dl Ge ee kb be n lb See E vas umes 26 evil kab DD rgrbrr o o TD D DZDZi m F_m apo i 35 Working with Large Pil 5s u nan era S yt aba r et kk a Ree jek e ek N aeons 40 Da ieee gt pasate tact rr E teas leases A T AET AE EAE ENA ETA E E E T 44 5 SVEM MINTEN C E nsan a AEA 45 Pay Man ayn an E E E E E enue Enea Deuaee Sra 46 Monthly Birane nr a E A E n 47 Perodie Mame nd 166 rresia nee E E E A y n A E EE N 49 Appendix Ax Ordering INtor maton eressero E A 50 Appendix B Performance TrackIing ccccscceceececeeeeceeeeceeeesseeeseeaeseeeeseeeeseseeseees 52 Pertormance Trac kine yyyyyyyzxxx pgg E9x xa oexMxNDPD xg g oxoi 53 Baseline Calc tl atl Onis ys yy Suey be n an y N ye we tub N wel yen ban s o edab 55 Perhorimiance Vest yyy 2oxoonDmmmmmmmnnDnDnDDDgxa2222oox i a o cmnoaoanypgp000000eeemWmLT 59 REPOT p re A ar rare e ee o wrrwwo JJJrr__i 63 Appendix C Instrument Description cccccececceceececeececeececeecuseeaesecaeseeeeseceeees 68 Dy SLCIA OI PON I rrr E rk 68 DY eNO PECICA S sraa e gg2 2 yrrr 20 69 Operation Principlesand Technical Cyr view c ye c kad ba 2a i S N VA NAN N 70 UA AICS ies Act pica 0 E te a ached a ce A en A
45. M New Experiment No template No template No template No template defined defined defined defined Click here to select one Click here to select one Click here to select one Click here to select one Blank Tube Experiment Blank Plate Experiment None None None None No template No template No template No template No template defined defined defined defined defined Click here to select one Click here to select one Click here to select one Click here to select one Click here to select one oS Functions amp jan os Cy ic n M h Note Results of instrument performance tracking tests are available for all users under the Reports option on the Performance Tracking menu but the Baseline Calculations and Performance Test functions can only be performed by authorized users Attune Acoustic Focusing Cytometer User Guide 63 Attune The sample report below shows the Blue Violet configuration with the Performance parameters FSC SSC BL1 BL2 BL3 VL1 VL2 and VL3 The Blue Red Report Example configuration parameters are FSC SSC BL1 BL2 BL3 BL4 RL1 and RL2 Performance Report Instrument Name Your Instrument Name Operator Administrator Serial Number Your Instrument Serial Number Run Date 11 15 2010 3 41 56PM Institution Your Institution Name Product Name 4 Peak Beads Software Version 1 2 Lot Number 756080 Laser Delay 312 Expiration Date 02 11 2013 Detector Settings Quant
46. Next Sample gt L R IM Compensation cores 7 i j t 4 12 27 2011 05 53 AM 1 Other Options i FSC H 10 6 Global Workspace Display 10000 events i Global Instrument Settings Sample Dispensed Se IU Compensation A1 Gate Ungeted js as dane Acquisition Volume 50 yl 170 pl total draw volume Experiment Name New Experiment 4 05 15 2012 1117 AM Mon ety ET a ee I w 25 ul min Name Evert Count Parent a com lw z oO Everts 40 405 190 000 Ji Unstained Control ane Ox 39 980 WH SHE J s J B12 J 5L3 Click on edge and drag to resize vu ji ve kv IF Collection Panel Cr instrument Configuration mm Specim i 2 RT Cee Morepa Default Instrument n KE AEEA 112 Attune Acoustic Focusing Cytometer User Guide Customizing the Workspace Working with e Click and drag objects to move them in the Workspace Workspace Objects e To resize an object click on its edge and then drag the edge to resize e To select multiple objects click on the first object press CTRL and then click on another object Selected objects are enclosed by a blue border Scatter Tube Blue Laser Tube The selected Tube has no data i Parent gate has no data zi i F H 10 6 BL1 H e To delete an object such as a chart from the Workspace click to select and then press Delete e To undo or redo an action click on the appropriate icon from the Quick Access Toolbar see page 108 You can also use keyboard sh
47. P e Shutdown is an automated function that initiates the cleaning cycle and post cleaning rinse This mode requires user supplied bleach Attune Wash Solution and Attune Shutdown Fluid e Startup is an automated function that starts the fluidics optics and electronics of the Attune Acoustic Focusing Cytometer The Startup functions include priming the instrument fluidics and allowing the laser time to warm e Stop is used to end all data collection e Clear is used to delete the data from the screen It refreshes the Workspace while the instrument is in Run or Record mode You can initiate some of the fluidics functions by clicking Functions in the Instrument Status Bar and then selecting the appropriate function from the menu items Shutdown can only be run from the Login Screen De bubble Wash Unclog Rinse Startup Shop Clear Error Functions Q IMPORTANT The lasers are powered down during the Wash and Shutdown cycles The lasers must warm up for at least 10 minutes before running additional samples Attune Acoustic Focusing Cytometer User Guide Appendix E Troubleshooting This section includes the following topics e Tips to help you troubleshoot your experiment e Technical Assistance Information Note For Software Troubleshooting refer to the Attune Cytometric Software Release Notes or contact customer support Attune Acoustic Focusing Cytometer User Guide 139 Cytometer Troub
48. USER GUIDE applied biosystems vy kila technologies Attune Acoustic Focusing Cytometer USER GUIDE Publication Part Number 4453328 Rev F Revision Date June 2012 technologies Copyright 2012 Life Technologies Corporation All rights reserved For Research Use Only Not intended for any animal or human therapeutic or diagnostic use Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document LIFE TECHNOLOGIES AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF Limited Use Label License Research Use Only The purchase of this product conveys to the purchaser the limited non transferable right to use the product only to perform internal research for the sole benefit of the purchaser No right to resell this product or any of its components is conveyed expressly by implication or by estoppel This product is for internal research purposes only and is not for use in com
49. a aa ae d A WR d apa l le z Ken 140 Performance Tracking T1rouDleslhOOliTI ic 4 5 sekt5 i24y55 y0155k0i nein 44135514 5de babek an s A d e eee nates 144 Sampl Troubleshoo LING J 5yan n Ken GANA Pal k A te a ke a i e A Kal n ande k 145 Appendix F Limited Product Warranty ccccccccecececeececeeeeceeeeseceeseseeseeaeees 146 Appendix Sar LV ose xelan n n San KEM b ke CE N n ben a beka SE DES Arada ew KER a KEN ye ketan 148 Safety Conventions Used in this DOcument Ei eee kereke kerek ke keke keke kre Ke KAK AKAR AR 149 SymbDols or TASTE UTM ONES u ana le kk k NN Maka k eh A N Pandan all Hek N tsa ance N Mek nS ARA 150 Dalety Labels S l alal P N ll A o 22 2d2 152 Gremeral in set w ll SNN reee rr e a 153 anka 222mmnnnnn2H HHHjjrrrhrhrrr 154 Chemica Waste O TO y lu le kane ek y ya ek ek Wara e Ne w b e an e ek d k be a 155 Bire nl l Da DD A T R AR E 156 Physical Hazard Safety us klan See N N e KE E sla wseostasaer au ANN ke an n AN A eva Va eee net Uae ee e A 157 BIOlOSICA ELA Zel QZ rrr rrr a aE Sa 157 Eiser ALL SEY cas NyyvrrJrrrrrr c r r r _ r rlrll rrr 158 Safety and Electromagnetic Compatibility EMC Standards cece eee keke kee 159 DS E E e NA y na E SE A A CN y r oases E a vada Aegean ee KA 160 Attune Acoustic Focusing Cytometer User Guide About t
50. age 44 5 Tochange the Parameter and or the Scale right click directly on the plot axis Red Laser Sample 200 7 Time FSC A 150 SSC A BL1 A BL2 A 50 Cut Ctrl x Copy Ctrl C Delete Del Duplicate Save As Scale b Customize Attune Acoustic Focusing Cytometer User Guide 19 Using a Pre Populated Template New Experiment from the Experiment Explorer 20 1 From the Main Menu screen double click Template you want to use New Experiment Pre populated Workspace opens and the Experiment Explorer displays the new Experiment created from the template Alternatively from Experiment Explorer right click My Experiments and choose New Experiment Using Template N Note You can customize any of the objects displayed on the Work Area by right clicking on the object To change the parameters and or the scale right click directly on the axis 1 From Experiment Explorer right click on New Tube Experiment the My Experiments then select New Tube New Tube Experiment using Template Experiment New Plate Experiment New Plate Experiment using Template New Folder Import Tube Experiment Import Plate Experiment Open My Experiments Folder 2 Enter the experiment name and the number of specimens and samples desired The default Workspace and default Instrument Settings are selected Confirm that the settings are correct for your experiment or change as requi
51. aid samples click the trash icon W Note If you disable all available sample overlays the display mode will default to Display active tube only When you re enable the overlay samples you must change the display mode back to Display selected tubes below only or Display active tube and selected tubes below only Attune Acoustic Focusing Cytometer User Guide 117 Gates Tab In the Gates tab you can add gates to the selected plot and manage them Derived Gates can only be managed via this tab and can be accessed from any plot Customize You can use the following options to customize workspace items General XAxis YAxis Samples Gates Back Gates You can add a gate to any plot using the toolbar or context menu Histogram gates are only valid for histogram plots and the other gate types are only valid for dot or density plots Manage your gates ow Available Gates M El R M o R2 e To show or hide a gate from the plot click the box next to the desired gate e To remove a gate from the plot click the trash icon W Back Gates Tab You can use the Back Gates to display descendent gate data on a plot Customize Customize You can use the following options to customize workspace items E General XAxis Y Axis Samples Gates Back Gates You can add a back gate to a dot plot using the context menu Gated plots can only display back gates from descenda
52. al ga Find Expiration Date 05 20 2021 Curren t Baseline 4 Baseline 5 17 2012 3 33 PM 756080 Levey Jennings Report v Daily 5 17 2012 3 38 PM Instrument Name Operator Administrator Previous Baselines Serial Number NA Run Date 5 17 2012 3 38 PM Baseline 12 14 2011 9 23 AM 756080 Institution Product Name 4 Peak Beads Software Version 2 1 0 7869 LotNumber 756080 Expiration Date 5 20 2021 FSC Delta PMT Voltage FSC Bright Bead HPCV o0 4 50 75 2 25 50 0 00 25 775 5 50 y 25 e 25 00 50 25 75 50 100 75 A A SSC Delta PMT Voltage SSC Bright Bead HPCV 75 10 25 50 8 00 25 5 75 0 3 50 1 25 23 e 1 00 e 3 25 75 5 51 100 FN 125 10 00 x Kv Criteria Met Borderline Out Of Range No Data Page 1 of 4 5 17 2012 3 38 PM e Criteria Met If the daily performance of the particular detector is within the limits set by the baseline calculation it is plotted as a solid green circle in the Levey Jennings chart e Borderline If the daily performance of the particular detector deviates slightly from the baseline it is plotted as a solid yellow circle in the Levey Jennings chart e Out of Range If the daily performance of the particular detector deviates significantly from the baseline it is plotted as a solid red circle in the Levey Jennings chart e No Data If the daily performance test is unable to find a PMT or HPCV it will be indicated in the Levey Jennings chart with a solid w
53. ameters Threshold Voltage Custom Parameters Advanced Options Please enter a threshold x1000 for each parameter Use the options in the corresponding drop down list to customize your threshold Please enter a threshold x1000 for each parameter Use the options in the corresponding drop down list to customize your threshold FSC SSC BLi 5 FSC ssc BL1 BL2 BL3 BL4 RLI RL2 hoo o o j 10 o L _ oo 10 10 ko 10 10 10 10 Cand cI cI Lr Ca Or v Ignore v Ignore v Ignore vj Ignore w Ignore v Ignore vi Ignore z or Ignore w Ignore w Ignore w Ignore w Ignore w Ignore w Ignore iw xan a You can also combine all thresholds using Boolean operators If the And operator is selected all the thresholds set will have to be met before the data is collected If the Or operator is selected data collection begins when at least one of the thresholds is met If the Ignore operator is selected the detector is ignored for thresholding purposes Voltage Tab Voltage tab allows you to select the voltage for each detector Instrument Settings Experiment 06 16 2010 02 36 PM Instrument Settings Experiment 07 01 2011 09 34 PM instrument Settings Instrument Settings You can use the following options to update instrument settings You can use the following options to update instrument settings
54. ample collection rate Q IMPORTANT We recommend that you sanitize the system between users See Inter User Wash Procedure on page 17 Shutdown Cycle The Shutdown cycle is performed at the end of each day to ensure that all sample fluid and dyes have been removed from the system and that the fluidic lines and two pumps are filled with Attune Shutdown Solution to prevent build up of salt deposits and cellular debris Automatic Shutdown can take up to 30 minutes Attune Acoustic Focusing Cytometer User Guide 137 Fluidics Functions 138 In addition to the Modes of Operation described on the previous page the Attune Cytometric Software controls the fluidics functions of the Attune Acoustic Focusing Cytometer These functions are e De bubble is a user initiated function for clearing bubbles in the fluidics lines of the cytometer e Wash is a user initiated system cleaning between sticky samples This function requires user supplied 10 bleach solution A IMPORTANT We recommend that you sanitize the system between users See Inter User Wash Procedure on page 17 e Unclog function is a user initiated back flush operation to remove clogs from the sample probe and flow cell e Rinse automatically flushes the system between samples to minimize carryover This function can also be user initiated During the rinse the Red Status Indicator Light will stop blinking when it is okay to load a new sample onto the SI
55. an overlap between the wavelength ranges Compensation is the mathematical method used to correct the overlap of one fluorophore s emission into another fluorophore s emission channel The Attune Cytometric Software calculates the compensation settings automatically as it guides you through the process Q IMPORTANT Once you click Record on any of the compensation controls within the compensation setup the Instrument Settings for all fluorescent channels will be shaded gray and cannot be changed It is critical that you optimize voltages prior to recording any sample or any compensation controls Compensation 1 Select Compensation from the main menu then select Compensation Setup Setup Alternatively double click Compensation or right click and select Compensation Compensation Setup under the same folder that contains your optimized Experiment in the Experiment Explorer Compensation Matrix Mal v m Remove Compensation Compensation Setup dialog box opens The control samples vary by configuration The Blue Violet configuration control samples are Unstained Control VL1 VL2 VL3 BL1 BL2 and BL3 The Blue Red configuration control samples are Unstained Control BL1 BL2 BL3 BL4 RL1 and RL2 Compensation Setup L Compensation Setup Compensation Setup Compensation Setup Use the following options to set up compensation Use the following options to set up compensation Compensate on
56. ance test results are shown as Out of Range along with the date and time of the run e The Performance Test Report is saved as a PDF file You can use the toolbar above the report to print the report or save it on a location of your choice e g flash drive B LJ ira IN QO ioo e coborat Aone ef ey frre CC gt Attune Acoustic Focusing Cytometer User Guide 61 Levey Jennings Report 62 The results of the performance test can also be viewed as Levey Jennings charts which provide a visual indication of the performance of the cytometer over time To review the Levey Jennings Report click Levey Jennings Report tab on the upper left corner of the Performance Test screen Performance Test Report Levey Jennings Report A8 amp applied A biosystems Current Baseline 6 9 2010 756080 Lot No 756080 Expiration Date 02 11 2013 Perfo ce Test Report Levey Jennings Ret RQ im ne 4 23 Current Baseline Baseline 6 9 2010 756080 V Dally 6 9 2010 3 35 PM Performance Report The Levey Jennings Report tracks the HPCV and changes in PMT voltages for all channels to check for shifts and trends in cytometer performance RS ews Attune Cytometric Software v2 1 Admin hoo A8 Pyat 3 biosystems ax by Kafe technologies Current Baseline 5 17 2012 3 33 PM 756080 Performance Test Report Levey Jennings Report Lot No 756080 bi Page 14 gt bl QQ GnePage
57. arameters Defining Custom Parameters Setting Threshold Setting Voltage values and other Advanced Options a Compensation opens the Compensation Setup window Specimen contains a list of the Samples to be analyzed Sample opens a Workspace local or global and instrument settings local or global The active sample is indicated by a Visual cue viL e A Sample with no data to be recorded is represented by a tube icon e lt A Sample with recorded data is represented by a tube icon overlaid with a disk Folders are used to organize your Experiments Each Folders icon can contain multiple Experiments and other folders Visual cue indicates the active Sample It is located on the left side of the Experiment Explorer If the active Sample is off screen the visual cue indicates which direction up down right in the explorer tree the Sample is located Clicking on the indicator automatically brings the active Sample into view on the explorer tree Attune Acoustic Focusing Cytometer User Guide 101 Right Click on Sample Wi Right Click Options in the Experiment Explorer Open Sample provide a number of functions that are specific Sai i to the object in the explorer tree View FCS File e Sample level right click allows you to R FCS File s 1 Van manage FCS files manage sample level Export FCS File s Instrument Settings and Workspaces manage Samples and view Sample Export Statistics j Print Propert
58. ates e Performance Tracking e Reports The administrator can also designate the Institution name Instrument name and set the Instrument Serial Number Assigning Data 1 Click Options La shortcut button in the Quick Access toolbar Alternatively Attune Acoustic Focusing Cytometer User Guide 133 Storage Paths click Options under Global in the Home tab Only a user with Administrator privileges can assign data storage paths Options window opens 2 In the Options window click Administrator 3 Assign the appropriate path using the Browse function Q IMPORTANT When reassigning data storage paths the administrator must copy the existing files from the original location to the new one Adding Users 1 Click Options E shortcut button in the Quick Access toolbar Alternatively click Options under Global in the Home contextual tab Only a user with Administrator privileges can add new users and assign user privileges 2 Click User Management Options Options You can use this to manage users and general application settings General a Sy Manage users working with Attune Cytometric Software Administrator User Management User Name Full Name Phone Number Styles Admin Administrator Mike Mike Doe 3 Select New User User Use the following options to set user information and access privileges User Name Mike Full Name Mike Doe Phone Number
59. by organizing control elements in logical groups under contextual tabs Each tab relates to a type of activity The ribbon tabs are the Home Insert Compensation View and Plate and they contain common commands that apply only to the selected item Objects that are not available as a function in a particular context are shaded gray in the tab y Note The Plate tab is for use with the optional Applied Biosystems Attune Auto Sampler Refer to the Applied Biosystems Attune Auto Sampler User Guide Part no 4479066 and the Applied Biosystems Attune Auto Sampler Quick Reference Guide Part no 4479067 for instructions Home The Home tab provides editing and formatting functions for the Workspace and is organized into five sections Clipboard Font Editing and Global Compensation View Plate Select All T sall Ir e Load Events Unselect A ara ease ee a 3 All Events B i U Aw Sr Delete Spans host zir na Fort Editing Size and Position Global Analysis e Clipboard allows you to cut copy and paste selected objects and text in the work area It also contains Undo E and Redo buttons e Font allows you to pick the font size style color and background color used for the selected object in the work area e Editing contains shortcuts for selecting unselecting and deleting any of the objects in the work area e Global contains the shortcuts to Options Main Menu and Transaction Log QV
60. categorized as a Class I laser product When safety interlocks are disabled during certain servicing procedures and or input output optics covers are removed the laser can cause permanent eye damage and therefore is classified under those conditions as a Class 3B laser Laser Safety To ensure safe laser operation Requirements e The system must be installed and maintained by a Life Technologies Technical Representative e Allinstrument panels must be in place on the instrument while the instrument is operating When all panels are installed there is no detectable radiation present If any panel is removed when the laser is operating you may be exposed to laser emissions in excess of the Class 3B rating e Do not remove safety labels Additional Laser Refer to the user documentation provided with the laser for additional Safety Information information on government and industry safety regulations WARNING LASER HAZARD Lasers can burn the retina causing permanent blind spots Never look directly into the laser beam Remove jewelry and other items that can reflect the beam into your eyes Do not remove the instrument top or front panels Wear proper eye protection and post a laser warning sign at the entrance to the laboratory if the top or front panels are removed for service 2 WARNING LASER HAZARD An overheated laser can cause severe burns if it comes in contact with the skin DO NOT operate the laser when it cannot be
61. consumable provided however that the warranty will not last for more than thirty 30 days after Customer opens consumable s original container Life Technologies does not warrant that the operation of the instrument or its operating software will be uninterrupted or error free Warranty claims must be made within the applicable warranty period Warranty exceptions The above warranties do not apply to defects resulting from misuse neglect or accident including without limitation operation with incompatible solvents or samples in the system operation outside of the specifications or not in conformance with the instructions use in combination with software or products not supplied or authorized by Life Technologies modification or repair of the product not authorized by Life Technologies relocation or movement of the instrument by Customer or any third party not acting on behalf of Life Technologies or intrusive activity including without limitation computer viruses hackers or other unauthorized interactions with instrument or software that detrimentally affects normal operations Without limiting the above mentioned computer hardware monitors accessories software or other products not purchased from or supplied by Life Technologies Non LIFE Product are not covered under the foregoing warranty even if such Non LIFE Product is integral to functional use of a Life Technologies product 146 Attune Acoustic Focusing Cytometer User Guid
62. cooled by its cooling fan Always wear appropriate laser safety goggles 158 Attune Acoustic Focusing Cytometer User Guide Safety and Electromagnetic Compatibility EMC Standards U S and Canadian Safety Standards Canadian EMC Standard European Safety and EMC Standards Australian EMC standards This section provides information on e U S and Canadian safety standards e Canadian EMC standard e European safety and EMC standards e Australian EMC standards The Attune Acoustic Focusing Cytometer has been tested to and complies with standard UL 61010 1 CSA C22 2 No 61010 1 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements FDA Radiation Control for Health and Safety Act of 1968 Performance Standard 21 CFR 1040 10 and 1040 11 as applicable This instrument has been tested to and complies with ICES 001 Issue 3 Industrial Scientific and Medical Radio Frequency Generators Safety This instrument meets European requirements for safety Low Voltage Directive 2006 95 EC This instrument has been tested to and complies with standards EN 61010 1 2006 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements EN 60825 1 Radiation Safety of Laser Products Equipment Classification Requirements and User Guide EMC This inst
63. correctly Use positive control to set PMT voltage threshold etc Reagent has degraded Restain sample with fresh reagent Primary antibody is not compatible with the secondary antibody Ensure that the secondary antibody was raised against the species in which the primary antibody was raised Lasers are not aligned properly Re run the Performance Test if it fails contact Service High fluorescence from the sample Antibody concentration is too high Reduce the amount of antibody added to the sample Excess antibody is trapped inside the cell Ensure adequate washing of the sample with wash buffer containing permeabilization reagent Inadequate blocking of the sample Perform the blocking step prior to staining the cells Experiment is not optimized correctly Readjust PMT settings to ensure that all populations are on scale Two or more cell populations are observed when there should be only one Inaccurate gating Revise gate to only include the population of interest Target protein is expressed on multiple cells Verify the expression level and ensure adequate cell identification and separation Cell doublets e Filter cells to remove clumps e Dilute the sample to reduce coincidence Non specific staining due to dead cells Use an appropriate dead cell stain to eliminate dead or dying cells Attune Acoustic Focusing Cytometer User Gu
64. cusing Cytometers and emit fluorescence signals at designed levels to all the channels in the cytometer Each vial of performance tracking beads contains a mixture of four beads at an equal concentration that differ in relative fluorescence emission intensity blank dim medium and bright The blank beads in Attune Performance Tracking Beads have a nominal diameter of 2 4 um The dim medium and bright intensity beads have a nominal diameter of 3 2 um Q IMPORTANT Prepare the Attune Performance Tracking Bead suspension immediately before use Label 12 x 75 mm or 1 5 mL microcentrifuge tubes Mix the contents of the bead vial by gentle inversion or gentle vortexing 4 To prepare the tracking bead suspension for Baseline Calculations add 4 mL of Attune Focusing Fluid or phosphate buffered saline PBS and 3 to 4 drops of Attune Performance Tracking Beads into the labeled tube For daily Performance Test 1 drop of Attune Performance Tracking Beads in 1 mL of Attune Focusing Fluid or PBS is sufficient Mix the bead suspension by gentle inversion or vortexing Use the beads immediately after mixing or store the diluted bead suspension at 2 6 C protected from light for no more than 4 hours WARNING Attune Performance Tracking Beads are non hazardous and may be disposed according to local regulations 1 Before running a new lot of beads go to www lifetechnologies com navigate to the Attu
65. d data The Attune Cytometric Software has two modes for data collection the Run mode and the Record mode ys Note If you wish to collect more 500 000 events you must limit the number of acquisition parameters to 10 or fewer To select or de select parameters for acquisition open the Instrument Settings from the View tab see Parameters Tab page 109 You will be prompted if more than 500 000 events are entered without limiting the parameters to 10 or fewer Attune Cytometric Software In order to collect greater than 500 000 events you must have 10 or less parameters selected Select OK to continue with the event limit defaulted to 500 000 axan Gs Run Mode The Run mode is initiated when Run on the Collection Panel is selected In this mode the software starts data acquisition but no FCS file containing the data is created e This mode is primarily used for adjusting the various parameters for cytometer and experiment optimization and customizing the charts for recording e The software actively stores only 20 000 events and once that limit is reached it recycles the events e During the run the events are displayed on the plots as the graphs are being populated ys Note If compensation is available and turned ON data will be display compensated during acquisition Record Mode The Record mode is initiated when Record on the Collection Panel is selected During recording
66. dd and remove compensation controls by opening the Compensation Setup and selecting deselecting channels 3 The Compensation Setup Guide directs you through the steps necessary for calculating compensation for your samples Compensation Setup Guide Import FCS file Show Compensation Setup Load Unstained Control Click Run Click Record Position Gate 7 Do not show this dialog again Close 4 Install the tube containing the unstained control beads cells on the sample injection port as prompted by the software 5 Push up the tube loader to the active position in the sample injection port and click Run on the Collection Panel Attune Acoustic Focusing Cytometer User Guide 27 28 6 Wait until the sample equilibrates and click Record 7 Repeat the process for each of the single stained controls making sure that the positive signal for all samples is on scale Scatter SSC A 10 6 T T T T T IT 5 6 7 8 104 10 0 1 2 3 lt FSC A 10 6 CD3 FITC BL1 A 1 To view the Compensation Matrix select Compensation from the main menu then select Compensation Matrix The Blue Violet configuration shows VL1 VL2 VL3 BL1 BL2 and BL3 The Blue Red configuration shows BL1 BL2 BL3 BL4 RL1 and RL2 Compensation Matrix Compensation Matrix You can view manually edit or reset compensation matrix values here EZE
67. de Part no Applied Biosystems Attune Acoustic Focusing Cytometer Quick 4453802 Reference Guide Applied Biosystems Attune Acoustic Focusing Cytometer User Guide 4453328 Applied Biosystems Attune Auto Sampler Quick Reference Guide 4479067 optional Applied Biosystems Attune Auto Sampler User Guide optional 4479066 Additional resources are available on the Flow Cytometry Technical Resources page Go to www lifetechnologies com then search for Flow Cytometry to open this page There you can find protocols application notes and tutorials Attune Acoustic Focusing Cytometer User Guide Safety Information Note See Appendix C Safety on page 68 for the complete the chemical or instrument safety information Safety Alert Words Four safety alert words appear in Life Technologies user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below IMPORTANT Provides information that is necessary for proper instrument operation accurate installation or safe use of a chemical CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in
68. death or serious injury DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations gt eS Except for IMPORTANT safety alerts each safety alert word in a Life Technologies document appears with an open triangle figure that contains a hazard symbol These hazard symbols are identical to the hazard symbols that are affixed to Applied Biosystems instruments see Safety Symbols on page 151 SDSs The Safety Data Sheets SDSs for any chemicals supplied by Life Technologies are available to you free 24 hours a day For instructions on obtaining SDSs see SDSs on page 160 Q IMPORTANT For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer Attune Acoustic Focusing Cytometer User Guide 7 Attune Acoustic Focusing Cytometer User Guide 1 Daily Routine Workflow Before You Begin Startup Check Fluid and Waste Levels Fill the Fluid Tanks Power On the Cytometer and Computer Launch the Software and Run Startup Function Run Samples See Chapter 2 Shutdown Check Fluid and Waste Levels Run Shutdown Function Q IMPORTANT Although the daily Startup and Shutdown procedures are automated and require minimal user input we recommend that you familiarize yourself with the instrument its operating principles and the software user inte
69. e Warranty limitations THE WARRANTIES IDENTIFIED ABOVE ARE LIFE TECHNOLOGIES SOLE AND EXCLUSIVE WARRANTIES WITH RESPECT TO SUCH PRODUCTS AND ARE IN LIEU OF ALL OTHER WARRANTIES EXPRESS OR IMPLIED ALL OF WHICH OTHER WARRANTIES ARE EXPRESSLY DISCLAIMED INCLUDING WITHOUT LIMITATION ANY IMPLIED WARRANTY OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE NON INFRINGEMENT OR REGARDING RESULTS OBTAINED THROUGH THE USE OF ANY PRODUCT INCLUDING WITHOUT LIMITATION ANY CLAIM OF INACCURATE OR INCOMPLETE RESULTS WHETHER ARISING FROM A STATUTE OR OTHERWISE IN LAW OR FROM A COURSE OF DEALING OR USAGE OF TRADE WITHOUT LIMITING THE FOREGOING IN NO EVENT SHALL LIFE TECHNOLOGIES BE LIABLE FOR CONSEQUENTIAL INDIRECT PUNITIVE INCIDENTAL OR OTHER SPECIAL DAMAGES SUSTAINED BY THE BUYER OR ANY OTHER PERSON OR ENTITY WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT LIFE TECHNOLOGIES IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES INCLUDING WITHOUT LIMITATION DAMAGES ARISING FROM OR RELATED TO LOSS OF USE LOSS OF DATA OR FOR LOSS OF REVENUE OR OTHER FINANCIAL LOSS NO EMPLOYEE OR REPRESENTATIVE OF LIFE TECHNOLOGIES HAS ANY AUTHORITY TO MODIFY THE TERMS OF THIS LIMITED WARRANTY STATEMENT AND ANY SUCH MODIFICATION MADE BY ANY EMPLOYEE OR REPRESENTATIVE OF LIFE TECHNOLOGIES WILL NOT BE BINDING ON LIFE TECHNOLOGIES UNLESS IN A WRITING SIGNED BY AN EXECUTIVE OFFICER OF LIFE TECHNOLOGIES THIS WARRANTY IS LIMITED TO THE BUYER OF THE PRODUCT FROM LIFE TECHNOLOGIES
70. e software OAB Home Insert Compensation View Plate ja i Load Events eee a Ep SS AllEvents Compensation Compensation Setup Compensation Setup Guide Matrix Compensation Analysis Refer to Calculate Compensation on page 26 Attune Acoustic Focusing Cytometer User Guide 105 106 View The View tab facilitates the configuration of an experiment and allows you to view FCS file contents of the active sample modify instrument settings and customize the Workspace It consists of Experiment Workspace and Show Hide a Home Insert Compensation View e Ti gt 2 C i e J Collection Panel J J Reset Windows Layout Load Events 2 r S 4 Instrument Configuration v Set Print Area All Events v FCS Instrument Save As d Refresh Page Print Quick Print Save As File Settings Template All Setup Print Preview DF Plate Experiment Explorer WV Plate Layout Status Experiment Workspace Show Hide Analysis RE Note The Flow Cytometry Standard FCS is the standard format for flow cytometer data files and provides the specifications needed to completely describe the data sets within e Experiment allows you to view the FCS file for the active experiment modify instrument settings and save the experiment as a template e Workspace allows you to zoom in and out of a selected plot refresh all items in the Workspace change the Page Setup settings page size order orientation and margins and pr
71. e Red Other fluorescent reagents PO PRO 1 DyeCycle Violet Fixable Violet Dead Cell Stain CellTrace Violet Calcein Violet SYTOX Blue FxCycle Violet Click 1T Pacific Blue Fixable Aqua Dead Cell Stain F2N12S apoptotic Fixable Yellow Dead Cell Stain F2N12S live Calcein Fluo 3 Fluo 4 TO PRO 1 iodide CFSE GFP JC 1 Di0C2 3 SYTOX Green DyeCycle Green Rhodamine 123 YO PRO 1 iodide Fixable Green Dead Cell Stain Click i1T ALexa Fluor 488 PI Fura Red AM cell permeant DyeCycle Orange JC 1 Di0C2 3 pHrodo Phagocytosis Particale Labeling Kit SNARF low pH SYTOX Orange Dead cell stain JC 1 Di0C2 3 Fixable Red Dead Cell Stain 7 AAD SNARF high pH isomers SYTOX AADvanced Dead Cell Stain DyeCycle Ruby Vybrant Dye Cycle Ruby 83 84 Red 638 nm Instrument Default Filter Eoo oo Hore Configuration filter nm range nm P PE Alexa Fluor 750 780 60 750 810 Qdot 800 PE Cy 7 Allophycocyanin APC 660 20 A Alexa Fluor 647 APC Alexa Fluor 750 780 60 750 810 APC Cy 7 APC H7 Blue Red Blue Red Blue Red i Attune Acoustic Focusing Cytometer User Guide Other fluorescent reagents Vybrant Dye Cycle Ruby Fixable Far Red Dead Cell Stain Click iT Alexa Fluor 647 FxCycle Far Red SYTOX Red Dead cell stain TO PRO 3 Fixable Near IR Dead Cell Stain Vybrant Dye Cycle Ruby Overall Geometry
72. e concentration of your sample is gt 1 x 10 cells mL dilute it down prior to running it on the Attune cytometer e The maximum recommended sample concentration for the Standard 500 uL minute and 1 000 pL minute collection rates is 5 x 10 cells mL Perform the Startup procedures as detailed on page 11 Perform the Performance Test procedures as detailed on pages 59 61 and verify that the Attune Acoustic Focusing Cytometer is in good working order Check the optical layout of the cytometer to verify that the filters are appropriate for the sample type and fluorophores you are using Place your mouse over the icon in the lower left corner to verify the optical configuration Either Blue Violet or Red Blue displays Blue Violet Red Blue e e gt Qood If you need to change the optical configuration 1 Click Options La shortcut button in the Quick Access toolbar 2 Select Configuration then select the Laser Color Configuration Attune Acoustic Focusing Cytometer User Guide General Administrator User Management Styles Resources Attune Cytometric Software Laser Configuration has changed 2 You will be logged out and will need to log back in for the changes to take effect Select OK to continue or Cancel to return to Options dialog Click OK to continue Log in to the software Inter User Wash We recommend that you sanitize the system between users Procedure 1 At standard 1 0
73. e cytometer During this process the software measures and records the following observables in all fluorescence detectors e Voltage required to place the bright bead in the target channel e Change in PMT voltage APMT e HPCV percent half peak coefficient of variation of the bright bead Using assigned MESF molecule of equivalent soluble fluorophore values for each fluorescent bead the software calculates the following for each channel e Relative detector quantum efficiency rQ e Relative background level rB e Linear regression e Laser delay setting The results of the performance test can also be viewed as Levey Jennings charts which provide a visual to track the HPCV and changes in PMT voltage to check for shifts and trends in cytometer performance Q IMPORTANT We recommend that you run the Performance Test at least once per day when the cytometer is in use Running 1 Turn ON the Attune Acoustic Focusing Cytometer and the computer see Performance Test page 12 Log on to the Attune Cytometric Software Click Performance Test on the Main Menu Performance Test screen appears Current Baseline Previous Baselines Attune Acoustic Focusing Cytometer User Guide 59 4 Prepare the Attune Performance Tracking Bead suspension see page 54 5 Check the beadlot number of the beads you are using The beadlot number is the first six digits printed on the label Make sure that the beadlot
74. e pull tube lifter down to initiate a rinse to avoid contamination before collecting again To continue click OK lower the tube lifter and allow the Rinse to complete Attune Acoustic Focusing Cytometer User Guide 39 Working with Large Files Recording Large Files Loading Large Files 40 When recording files with greater than 250 000 events the Attune Cytometric Software does not automatically load the entire data set Instead only the first 20 000 events of the FCS file are loaded and The Sample icon in the Experiment Explorer indicates that the Sample H contains recorded data The F5 icon appears on all plots and statistics boxes indicating that the displayed data is based on 20K total events The statistics text is italicized indicating the values are based on 20K total events When opening files with greater than 250 000 events the Attune Cytometric Software does not automatically load the entire data set Instead only the first 20 000 events of the FCS file are loaded and The Workspace opens with all of the plots and statistics boxes F5 displaying the F5 icon The statistics text is italicized indicating the values are based on 20K total events a 2T a a Dn oO U n n a E Dn Note When working with large files it can take a considerable amount of time to load the data into the Workspace The preview expedites the process of setting gates inserting plots and tw
75. e status display and Daily Performance Test Report is displayed 60 Attune Acoustic Focusing Cytometer User Guide Daily Performance The result of the daily performance test is displayed at the end of the run Test Report If the run is successful the Daily Performance Test Report screen summarizes the results and compares them with the target values The sample report below shows the Blue Violet configuration with the parameters FSC SSC BL1 BL2 BL3 VL1 VL2 and VL3 The Blue Red configuration parameters are FSC SSC BL1 BL2 BL3 BL4 RL1 and RL2 WT OTETLT T Attune Cytometric Software v2 1 Admin oy ee gt applied 3 biosystems ax Current Baseline 5 17 2012 3 33 PM 756080 Performance Test Report Levey Jennings Report Lot No 756080 Page 1 1 Q 125 y et Expiration Date 05 20 2021 Current Baseline 4 Baseline 5 17 2012 3 33 PM 756080 Performance Report V Daily 5 17 2012 3 38 PM Previous Baselines laatrunserst NANE Operator Administrator Baseline 12 14 2011 9 23 AM 756080 Serial Number NA Run Date 5 17 2012 3 38 27PM Institution Product Name 4 Peak Beads Software Version 2 1 0 7869 Lot Number 756080 Laser Delay 433 Expiration Date 05 20 2021 Detector Settings Laser Parameter Brighit Bond Bima Bead all ver n me eae paces Lin Reg Result Target MFI Voltage iciency B in Reg esul rg Actual MFI Voltage HPCV Q Violet FSC 700000 703340 00 1075 OmV 440 N A NIA N A Cri
76. eaking compensation Calculating statistics is CPU intensive and slows down the refresh rate of the Workspace Attune Acoustic Focusing Cytometer User Guide Refreshing the Refreshing the Workspace loads the entire data set You can refresh Workspace Workspace objects globally all at once or only those plots that require a refresh e To refresh the Workspace globally press the F5 function key or select Refresh All from the View tab These actions refresh all Workspace objects Clicking a F5 icon will refresh all plots and statistics that have the F5 icon Note Whenever you move a gate create a daughter plot or perform an operation that affects the relationship between displayed and calculated data the F5 icon appears on the object affected by the upstream change If compensation is adjusted all plots will require a refresh J Ed Workspace before a Refresh jr aed EH di a a i Be b l L aE ziii a a L i La n n n F Fr j g i m u i E i as J ie iar a m a Attune Acoustic Focusing Cytometer User Guide 41 42 Workspace after a Global Refresh i TH ji Workspace with Objects Requiring a Refresh TIL bev all i i ll a ji a a FEE Eir bx mlg Ht Diah n n kam l m Attune Acoustic Focusing Cytometer User Guide EJ Workspace requiring a Global Refresh after Compensation Adjustment
77. ect signified by the Ii or H icon represents a sample Instrument settings and Workspace are indicated by bold font and an asterisk respectively Attune Acoustic Focusing Cytometer User Guide 95 Attune Desktop The Attune Desktop is where you can run samples generate data and analyze the results It consists of e Quick Access Toolbar e Ribbon tabs Home Insert Compensation View and Plate e Collection Panel e Instrument Controls panel e Plate Setup panel for use with the optional Attune Auto Sampler e Heat Map Setup panel for use with the optional Attune Auto Sampler e Instrument Status Bar e Workspace e Experiment Explorer m Load Events All Events Matrix Ribbon Analysis Experiment Explorer Scatter BL1 A BL2 A 4 My Experiments 0 ev sec 8 100 100 4 12 14 2011 09 27 AM rr 7 80 80 EQ Global Workspace 0 events P E 60 60 e Global Instrument Settings 0 2 4 3 3 4 ia Compensation lt g 3 aii me Unstained Control Stop recording when 20 20 jM 5L1 Any of these conditions are met 0 0 li 5L2 Z 10000 Events on All Events_ 012345678 10 10 104 10 09 10 10 10 jM 813 5 Min 0 Sec FSC A 10 6 BL1 A BL2 A vL1 4 000 ul Ma v 2 O Record continuously MA viz si BL3 A VL1 A os VL2 A b 12 26 2011 06 34 PM 4 12 27 2011 05 53 AM 80 80 80 g Global Workspace 60 60 60 Global Instrument Settings 40 40 8
78. ectrical shock can result from operating the Attune Acoustic Focusing Cytometer without its instrument panels in place Do not remove instrument panels High voltage contacts are exposed when instrument panels are removed from the instrument Fuses WARNING FIRE HAZARD For continued protection against the risk of fire replace fuses only with fuses of the type and rating specified for the instrument Power DANGER ELECTRICAL HAZARD Grounding circuit continuity is vital for the safe operation of equipment Never operate equipment with the grounding conductor disconnected DANGER ELECTRICAL HAZARD Use properly configured and approved line cords for the voltage supply in your facility DANGER ELECTRICAL HAZARD Plug the system into a properly grounded receptacle with adequate current capacity gt e e gt Overvoltage Rating The Applied Biosystems Attune Acoustic Focusing Cytometer has an installation overvoltage category of II and is classified as portable equipment 156 Attune Acoustic Focusing Cytometer User Guide Physical Hazard Safety Moving Parts A WARNING PHYSICAL INJURY HAZARD Moving parts can crush and cut Keep hands clear of moving parts while operating the instrument Disconnect power before servicing the instrument Biological Hazard Safety WARNING BIOHAZARD Biological samples such as tissues body fluids and blood of humans and other animals have the potential
79. eets SDSs See SDSs on page 160 CAUTION Using cleaning or decontamination methods other than those recommended by the manufacturer may compromise the safety or quality of the instrument Attune Acoustic Focusing Cytometer User Guide 153 Chemical Safety Chemical Hazard Warning General Safety Guidelines 154 WARNING CHEMICAL HAZARD Before handling any chemicals refer to the Safety Data Sheet SDS provided by the manufacturer and observe all relevant precautions WARNING CHEMICAL HAZARD All chemicals in the instrument including liquid in the lines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument AN WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles To minimize the hazards of chemicals e Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous ma
80. efined defined defined Dak Click here to select one Click here to select one Click here to select one Click here to select one Click here to select one Ar ra Workspace with a blank work area opens and the new experiment is displayed on the Experiment Explorer N kmh h y Note By default the new Experiment displayed on the Experiment Explorer is labeled with the date and time it is created and contains no Specimens or Samples O 6 l A N EZ To rename select the Experiment and then press F2 Or right click the Experiment and choose Rename Experiment 2 To add a Specimen to your experiment right click the experiment and choose New Specimen 3 To add a Sample to the Specimen right click the specimen and choose New Sample Q gt Note To rename Experiments Specimens and Samples right click and choose Rename Alternatively highlight and then press F2 You can also rename Experiments Specimens and Samples by right clicking and choosing Properties and changing the name in the dialog box that opens Attune Acoustic Focusing Cytometer User Guide 4 To insert a Plot to your Work Area select the Sample in the Experiment Explorer then choose the type of plot from the Insert tab Plot Plot Plot Histogram Dot Density Plots ys Note For an overview of different types of plots see Plots in Appendix B Technical Overview on p
81. ended Solutions Event rate is too low Threshold level is too high Lower the threshold level PMT voltage for the threshold parameter is set too low Set the PMT voltage higher for threshold Sample injection port SIP may be clogged Run Unclog function or run Wash cycle If the problem persists contact Technical Support Sample is not adequately mixed Mix the sample to suspend the cells Sample is too dilute Increase the sample flow rate Loose sample syringe Check the sample syringe for leaks and tighten if necessary Low event rate with high CV Bubbles in flow cell Run De bubble function Erratic event rate Partial clog in the flow cell Run Wash cycle If the problem persists contact Technical Support Sample has large clumps Filter the sample prior to loading to the instrument Loose syringe Check syringes for leaks tighten if necessary Focusing Fluid pump or Sample pump is not delivering the correct volume and or is operating at inaccurate speed Contact Technical Support Contaminated sample Prepare new sample using clean tubes Bubble in fluidics lines Run De bubble function Sample is not aspirating Loose sample syringe Check the sample syringe for leaks and tighten if necessary Defective sample syringe Replace sample syringe Pinch in valve tubing Contact Technical Support Clog in SIP tube
82. enus to customize the parameters for Menus an experiment The context menu that is displayed depends on where you click on the Plot Blue Laser Sample Red Laser Sample 150 SSC A a E BL1 A Cut Ctrl X 50 Copy Ctrl C Delete Del BL2 H Duplicate Save As BL4 H Statistics RL2 H Back Gates New Gate lt w N y ets Set Population b Right click on the inside BL3 W j Customize BL4 W to open this menu Cut Ctrl x Copy Ctrl C Delete Del Duplicate Save As Scale gt Customize Right click on the plot and select Customize to open the Customize Plot dialog box Customize Plot dialog box has six tabs General X Axis Y Axis Samples Gates and Back Gates Blue Laser Sample 10 Pi Cut Ctri X Copy Ctrl C Delete Del Duplicate Save As Statistics Back Gates r 6 New Gate b p Set Population Attune Acoustic Focusing Cytometer User Guide 115 General Tab General tab allows you to select the plot type and the density of the displayed events and enter the chart title It also allows you to select the text styles and the border used for the plot Title Customize Customize You can use the following options to customize workspace items Plat Type Dot Plot Density 100 p Chart Title Text Style Tahoma 075 SJ JE A gt Border width 0 Ba Include Name cf 1 El Experiment E Specimen Sa
83. er is used i e the filter has a pass band centered at 530 nm and the width of the pass band is 30 nm However it is impossible to choose filters which measure only one dye For instance the Alexa Fluor 488 dye has a significant emission in the region that RP E is measured 575 nm Therefore the emission from Alexa Fluor 488 dye will register in 530 nm and 575 nm bands If R PE is also present it will contribute to the 575 nm band Compensation is the mathematical process for correcting for the amount of the Alexa Fluor 488 dye fluorescence in the 575 nm band so that R PE fluorescence can be accurately measured Performing multicolor analyses can complicate this process further because fluorophores are not usually spectrally separated very well Fluorescence spill over can be estimated by running single fluorescence controls specific for a certain dye and then subtracting out the fluorescence in the other detection channels thus leaving the true signal of the other fluorophores If a fluorescent dye emission is collected through three different filters then the amount of fluorescence captured through the first filter can be estimated based on how much spillover or contaminating signal is present in the second or the third filters In the example above the Pacific Blue conjugate has some fluorescence in the 530 30 filter and very little in the 575 24 filter therefore the amount of compensation required in the 530 30 filter will be
84. ercent Total C Concentration ev ul TERE e Standard Deviation e Coefficient of Variation e Concentration ev pL Note You can change the default statistics using the Styles tab in the Options dialog box 130 Attune Acoustic Focusing Cytometer User Guide Managing Experiments Experiment The Experiment Explorer is used to manage experiments Each Experiment is Explorer associated with an Instrument Setting and a Workspace There can be more than one experiment in the Explorer each experiment can have more than one Specimen and each Specimen can have more than one Sample Each Sample is associated with its own FCS file Experiment Explorer il G My Experiments d 2 Experiment 1 Workspace gt Instrument Settings al Compensation al gg Specimen gt H Sample 1 Hi Sample 2 4C Experiment 2 Lal Workspace gt gt Instrument Settings im Compensation d e Specimen Hi Sample 3 HE Sample 4 3 Experiment Folder Attune Acoustic Focusing Cytometer User Guide In this example there are two Experiments Experiment 1 and Experiment 2 in the Experiment Explorer Experiment 1 and Experiment 2 have their unique Workspace and Instrument Settings Sample 1 has recorded data li while Samples 2 3 and 4 do not have data associated with them ii Sample 1 has its unique Instrument Settings as indicated by the bold Sample name i Sample An asterisk following the Sample name i Sample indicat
85. es longer wavelengths and transmits shorter wavelengths over the active range of the target spectrum usually the ultraviolet and visible region For example an SP 530 filter permits wavelengths shorter than 530 nm to pass while reflecting or absorbing wavelengths longer than 530 nm 460 nm 540 nm SP 530 woe Dichroic mirrors also called reflective thin film or interference filters are produced by coating a glass substrate with a series of optical coatings Dichroic filters usually reflect the unwanted portion of the light and transmit the remainder A dichroic filter is a very accurate color filter used to selectively pass light of a small range of colors while reflecting other colors By comparison dichroic mirrors and dichroic reflectors tend to be characterized by the color s of light that they reflect rather than the color s they pass Dichroic mirrors are essential to the optical path of a flow cytometer because they direct the emission light to the appropriate photomultiplier tube detector 540 nm 460 nm LP 530 Neutral density filters ND have constant attenuation across the range of visible wavelengths and are used to reduce the intensity of light by reflecting or absorbing a portion of it Attune Acoustic Focusing Cytometer User Guide 79 Compensation 80 Fluorophores emit light over a range of wavelengths Although optical filters limit the range of frequencies measured by a given detector
86. es resolution for up to 20 000 events per second Measurement The data is further processed by the field programmable gate array FPGA which simultaneously calculates pulse height area and width when the pulse exceeds the user specified threshold value width Pulse area Q D z 2 s g 9 trigger Be threshold front rear front extension extension Time 82 Attune Acoustic Focusing Cytometer User Guide Optical Layout Instrument Default Filter ENO on Configuration filter nm range nm P 450 40 430 470 522 31 907 537 603 48 079 627 530 30 919 945 574 26 061 587 PE PE Alexa Fluor 610 PerCP PE Alexa Fluor 700 PE Cy 5 5 TRI COLOR antibody PerCP Cy 5 5 PE Cy 7 PE Alexa Fluor 750 Qdot 655 Qdot 705 Qdot 800 Qdot 705 PerCP Pacific Blue Alexa Fluor 405 Blue Violet Qdot 525 Horizon V500 E CC LO O z S 2 gt Blue Violet Pacific Orange Qdot 605 Blue Violet Alexa Fluor 488 Fluorescein u C o a U CC 5 o C O O lt Q lt E o 40 N E aL QO lt b Cc o c O hot gt lt LLI O 2 LL v rn c Q D L v O c L O c D D ra v O c L O All Configurations Blue 488 nm 640LP gt 640 690 50 669 719 Attune Acoustic Focusing Cytometer User Guide Blue Violet TRI COLOR TC PE Cy 5 PerCP Cy5 5 PE Cy 5 5 Blu
87. es that the Workspace layout for that Sample has been modified and is unique to that Sample Sample 2 is associated with the default Instrument Settings as indicated by Sample name that is not bold i Sampie 2 Visual cue indicates that Sample 1 is active 131 Managing e Workspace layout and the Instrument Settings are the same for the various Experiments Specimens and Samples that are collected for a particular Experiment Therefore all Samples for Experiment 1 Sample 1 and Sample 2 in the example on the previous page have the same Workspace and Instrument Settings e However you may modify Instrument Settings and Workspace layout at the Sample specific level Sample specific Local Workspace and Instrument Settings are denoted by an asterisk and bold font respectively see previous page e Double clicking on Workspace opens the Experiment template which contains plots statistics text or images These plots will not be populated with any data until they are associated with a Sample e If the layout of the Workspace has been modified you will be prompted with the option to save the changes to the Workspace when attempting to open another Sample or Workspace from another Experiment Note By default all Samples share the same Workspace and Instrument Settings as their parent Experiment However when a new Sample is created the Workspace layout is copied to the Sample The Workspace template remains intact
88. federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the SDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS Handle chemical wastes in a fume hood After emptying the waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations If potentially hazardous waste is generated when you operate the instrument you must Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Attune Acoustic Focusing Cytometer User Guide 135 Electrical Safety AN DANGER ELECTRICAL SHOCK HAZARD Severe el
89. form baseline calculations and performance tests Attune Acoustic Focusing Cytometer User Guide Performance Tracking Performance Tracking is a comprehensive set of procedures to monitor the daily performance of the cytometer The performance tracking process involves e Running the same performance tracking bead particle set e Monitoring the changes in the coefficient of variation and the changes in PMT voltages e Tracking the linearity of the cytometer e Evaluating the detector quantum efficiency Q and optical background B Performance tracking is critical to ensure the accuracy and sensitivity of the cytometer and it provides information about the lasers and detection channels available on the Attune Acoustic Focusing Cytometer Maintenance on page 38 Note For optimal cytometer performance follow the periodic and scheduled laser and fluidics maintenance procedures see System Performance Tracking Functions i performance tracking through its Reports feature The Attune Cytometric Software provides automated Baseline Calculations and Performance Test functions with minimal user interaction and facilitates You can access these performance tracking functions from the Main Menu WT TEYETLT T Attune Cytometric Software v2 1 Admin applied A biosystems Performance Tracking eee Browse Experiments Last performance test completed on Wednesday Decembe
90. g through the optical cell the stream arrives at the waste container Laser Intercept Points Flow Cell Capillary Assembly Sample Loop Bubble Sensor Sample Tube Focusing Fluid l Reservoir Focusing Fluid Filter Focusing Fluid Movement via Sample Pump Movement via Focusing Pump Attune Acoustic Focusing Cytometer User Guide Acoustic Focusing Flow Rate The objective in flow cytometry is to measure the properties of individual particles as they move through the laser beam When a sample in solution is injected into a flow cytometer the cells or particles are randomly distributed in three dimensional space and must be ordered into a stream of single particles Acoustic focusing exploits the physical differences between cells or particles relative to the background carrier medium to position the particles or cells into a single focused line along the central axis of a flow channel independent of sample fluid flow In contrast to a conventional hydrodynamic sheath focused cytometer the Attune Acoustic Focusing Cytometer gives users tight control over the sample flow rate which in turn permits the control of interrogation time enabling higher sensitivity and precision The images below show the alignment and concentration effects of acoustic focusing on a whole blood sample Acoustic focusing off Acoustic focusing on Acoustic focusing on
91. gin the software directs you to the Main Menu screen IMPORTANT The default username and password for the Attune Cytometric Software are admin and password respectively After logging in assign a password to the administrator account as soon as possible To create additional users see Software Administration on page 133 Attune Acoustic Focusing Cytometer User Guide 93 Main Menu After you log in to the Attune Cytometric Software the Main Menu is displayed The Main Menu consists of three panels Performance Tracking New Experiment and Experiment Explorer i e Browse Experiments applied hesystems Performance Tracking k Browse Experiments a My Experiments a 12 14 2011 09 27 AM Workspace E Instrument Settings Performance Test Reports a Ji Compensation La Unstained Control of Last performance test completed on Wednesday December 14 2011 9 23 AM lk 8L1 lk BL2 New Experiment No template No template No template No template defined defined defined defined Click here to select one Click here to select one Click here to select one Click here to select one No template No template No template No template No template aie defined defined defined defined defined lo 7 j gt Click here to select one Click here to select one Click here to select one Click here to select one Click here to select one i 3 SE Fundtions A 4 New Experiment Pe
92. his Guide Audience Table of Acronyms This user guide is for laboratory staff operating maintaining and analyzing data using the Applied Biosystems Attune Acoustic Focusing Cytometer Q IMPORTANT For instructions on using the Applied Biosystems Attune Auto Sampler refer to the Applied Biosystems Attune Auto Sampler User Guide Part no 4479066 and the Applied Biosystems Attune Auto Sampler Quick Reference Guide Part no 4479067 The following table explains the acronyms used in the Applied Biosystems Attune Acoustic Focusing Cytometer User Guide Acronym ADC Br BL1 BL3 BL1I BL4 YoCV FSC YHPCV MESF MFI PMT PMTV RLI RL2 SD SIP SSC VL1 VL3 Definition Analog to Digital Converter Relative background level of detection channel Detectors which measure the output from the 488 nm laser blue on the blue violet configuration Detectors which measure the output from the 488 nm laser blue on the blue red configuration Percent coefficient of variation standard deviation mean x 100 It is a measure of variation in signal intensity generated as particles pass repeatedly through the laser beam and is expressed as a percentage of average signal intensity Forward scatter Percent half peak coefficient of variation Molecule of equivalent soluble fluorophore Mean Fluorescence Intensity as described by the mean ADC value for a given bead intensity population Phot
93. hite circle The Levey Jennings Report is saved as a PDF file You can use the toolbar above the report to print the report or save it on a location of your choice 4 EV j R Ik S e 100 dla Collaborate Az Sign 7 ey E bi Attune Acoustic Focusing Cytometer User Guide Reports You can access the archived results of the Baseline Calculations and Performance Tests to determine the daily performance of the Attune Acoustic Focusing Cytometer and monitor changes in its accuracy and sensitivity using the Reports function which is available through the Main Menu The performance tracking reports include a summary of baseline calculations review of the linearity of PMTs laser performance tracking and alignment as well as Levey Jennings plots that chart historical performance testing data See pages 64 65 for examples of Performance and Levey Jennings Reports hy Note By default the performance tracking reports are saved in C Program Files Applied Biosystems Attune Performance Tracking unless changed by the Administrator see Software Administration on page 133 wo WP FR Attune Cytometric Software v2 1 Admin oJ e applied biosystems Logout by Life technologies Performance Tracking Browse Experiments Last performance test completed on Wednesday December 14 2011 9 23 AM F Eue 12 14 2011 09 27 AM 12 27 2011 05 53 AM lC 12 27 2011 05 53 AM 1 05 17 2012 02 30 P
94. i cee iat eu Oita de a v n n te 72 FACOUSH se NN S N rr eeemnmnoog nryW neani 72 OPC S psec aes eos T S I 75 lser an S N gt rwrywywywvrvrzrvv ra r m_r_ aw 81 OPUN AYON DN Dr aE PO Pe nC re ore Poe Poree ero ee aera eee hare mere 83 INStrUMent EX ter Om COMI OMCI US yyy yk HE ND n InRHED rn nE nnmnmnoxxe n2xXc p p 86 Attune Acoustic Focusing Cytometer User Guide 3 Instrument IMtenlOrC OM BONIS xe eam ecient eer aa ear i ea 87 DUGLUS UNGICALOR Eie INS neesi ES S E E a 89 Instrument Reagents and Cons mableS siirsi aaa a a a a a R aka 90 Appendix D Attune Cytometric Software DeSCIription ccccececeeceeeceeeseeeeees 92 DOST CC EE E a Axay A E E E ane ee aa 93 Man Meierie DD ANEA EEN E N N ANE AEE 94 Aline Deskop YY a aa e T T ec a eee A ees 96 Mostru mernt SCUO S oenorseoni iraire n e A EAEE A A ERA E EAEE EAS AA A 109 Customize thie Athine Desktop oarnein r er AE E R 112 Customize TNE WOKS PaE rrean n n E T TO 113 CUSTOM ZI H A Pr xrtrrrrrrooooo_J_J w _e _eJvrJJ J rprrerrrr 115 CAUAS TOOLS errr r EER N E E ESRA r JJ 121 Warkino WIA otis S eoi a A E N a N N NE 127 Muna cil EPET C aaa E T 131 Wayla ln N NA N E T E At engiees 133 Modes OF Opera tt On es yak yay n y wal wes a E AEA 137 Appendix E TFOUDLESNOOLIN 0 s i 4 x y yy a reason in kb ay n eb heka n k A ka bb O KAN M Wa su kun er s y K k 139 Cytometer Troubleshoo Gne sesia a n ba e d l a Ek Wad
95. ia in the Collection Panel You can specify the collection criteria as Record continuously or set limits to collection by the number of events for specified gates total sample volume analyzed or by elapsed time see Collection Panel on page 97 Install the tube containing the sample on the sample injection port and lift up the tube loader to the active position Click Run The events are displayed on the plots as the graphs are being populated Wait a short time for the sample to equilibrate Q Important If you are collecting more than 500K events you should collect 10 or fewer parameters The progress shown is based on Event Max which is 500K for 11 or more parameters and 20M for 10 or fewer parameters Unless you set Collection Criteria to Record continuously the progress bar is displayed during collection 1 While still in the Run mode adjust the PMT voltages and Threshold values for the appropriate channels using the Instrument Configuration tab see Instrument Configuration on page 100 Make sure that the events are on scale Click Record to start data collection Data is recorded until any of the conditions set in Collection Criteria is satisfied Note Click Pause to temporarily halt the data collection You can click Run to resume the data collection from that point on Click Stop to stop the data collection The Attune Cytometric Software automatically saves the data in a uni
96. ide 145 Appendix F Limited Product Warranty Limited warranty Life Technologies warrants that all standard components of its Attune Acoustic Focusing Cytometer will be free of defects in materials and workmanship for a period of one 1 year after the date of installation and the software will be free of substantial programming errors or defects when properly installed However this warranty will not last longer than 15 months from the date of shipment Life Technologies also warrants that the instrument including software will perform in accordance with its published specifications when delivered Life Technologies will repair or replace at its discretion all defective components during this warranty period Life Technologies reserves the right to use new repaired or refurbished instruments or components for warranty service agreement replacements Repair or replacement of products or components that are under warranty does not extend the original warranty period Unless a different written warranty is included with product literature Life Technologies warrants that each consumable supplied with the instrument will meet its specifications stated in its published catalogs and any associated supplementary terms relating to the product This warranty lasts from the delivery of the consumable until either the consumable s expiry or use by date or if no expiry or use by date is specified for 12 months from the delivery of the
97. ies TE EAE roe e Specimen level right click allows you to Save Instrument Settings As Global create new samples manage specimens and apply the global Workspace to all Remove Local Workspace samples within a specimen Save Workspace As i wale AM e Workspace level right click allows you to Import Workspace open a Workspace save a Workspace and apply the global Workspace to all samples within the Experiment Duplicate Sample Delete Sample s Rename Sample e Right clicking on higher level objects i e Experiment provides functions to manage Sample Properties rr Right Click on Specimen a experiments New Sample Duplicate Specimen Delete Specimen Rename Specimen Export FCS File s Export Statistics Print Specimen Properties Right Click on Global Workspace Ga Open Workspace Save Workspace As NM ra to am wi ima Apply Global Workspace to All Samples withir Experiment F2 F2 is a windows shortcut to rename files Y Note A Sample name in bold indicates that the Sample H Sample has Sample specific instrument settings A Sample with an asterisk after its name has a Sample Hi Sample specific Workspace A Sample with a bold name and an asterisk indicates ie Sample that the Sample has Sample specific instrument settings and Workspace Sample Properties allow you to add notes to a sample 102 Attune Acoustic Focusing Cytometer User Guide
98. ight Bead HPCV Delta PMT mv ofo HPCY Page 3 of 4 VL2 Delta PMT Voltage VL2 Bright Bead HPCV 75 Delta PMT mv 0 HPCY VL3 Delta PMT Voltage VL3 Bright Bead HPCV 450 Delta PMT mY Page 4 of 4 66 Attune Acoustic Focusing Cytometer User Guide Attune Acoustic Focusing Cytometer User Guide 67 Appendix C Instrument Description System Components The Applied Biosystems Attune Acoustic Focusing Cytometer is shipped with the system components listed below All components are shipped at ambient temperature Component Quantity Applied Biosystems Attune Acoustic Focusing Cytometer 1 Either Blue Violet configuration Blue Red configuration Power cord kit universal voltage C13 2 5 m RC Cable USB 2 0 A B M M 6 ft RC Cable network RJ45 M M CAT6 STP 7 ft BLUE RC 24 inch monitor Dell computer including mouse keyboard and mouse pad Applied Biosystems Attune Cytometric Software CD Attune Performance Tracking Beads 3 mL Attune Wash Solution 500 mL Attune Focusing Fluid 6 x 1 L Attune 10X Shutdown Solution 250 mL Applied Biosystems Attune Acoustic Focusing Cytometer Quick Reference Card Applied Biosystems Attune Acoustic Focusing Cytometer User Guide 1 eje NOT Re Rt Rt SAIL Rt Rt Re w Intended Use For Research Use Only Not for use in diagnostic procedures 68 Attune Acoustic Focusing Cytometer User Guide
99. int the Workspace e While zoomed in on a plot you can toggle between plots using the Tab key left to right or Shift Tab keys right to left e Show Hide allows you to choose windows i e Collection Panel Instrument Configuration and Experiment Explorer to display or reset the windows layout You can hide the script dialog boxes to make the Workspace accessible for data analysis by un checking the Status box Print Area can also be turned on or off by clicking the checkbox Attune Acoustic Focusing Cytometer User Guide Instrument Status Instrument Status Bar indicates the status of the Attune Acoustic Focusing Bar Cytometer and displays cytometer errors as error indicator icons It also contains the Functions menu Experiment Summary and the Zoom tool Layout Default Instrument Settings Specimen Tube Functions 100 Scripts Status None Compensation None System Status Functions Experiment Zoom Status Indicators Menu Overview Tool Status Indicator Icons Startup successful and the cytometer is ready for operation System errors encountered low fluid levels pump timeout etc Startup required Cytometer connected but there may be a problem with running one of the cycles This warning often requires you to clear the error by selecting Clear Error from the Functions menu see below System detects problem with lasers Top cover is open A Fluid levels do not meet the required minimum
100. ion of interest so that the fluorescence histograms are reflective of the population for which you are optimizing your voltages Adjust the Fluorescence Channels to place your unlabeled sample in the appropriate area in the plot generally around 10 for unstained control Remove the unstained control from the sample injection port Fsc SSC BLI BL2 BL Vi V2 VL3 ee Se ee aT A M Ni coeton Panc g rerument cong rieirum ril Threshold x1000 Threshold x1000 Fn Ca a a si L soc 210c 220c 220c 255c sas rose 220d E E Ji ae Blue Violet Red Blue Attune Acoustic Focusing Cytometer User Guide Optimizing Instrument Settings for Single Stained Controls After you have optimized the instrument settings for Experiment Explorer 4x the unstained control optimize the instrument 4 ical controls for each of the single stained controls NENN 1 Stay on the Unstained compensation control 1 E 2 Install the first single stained control on the tube E Instrument Settings lifter For the Blue Violet configuration a H Compensation histograms are ordered as VL1 3 then BL1 3 For gt A unstained the Blue Red configuration the histograms are Aa vis ordered as BL1 4 then RL1 2 i v 2 3 Using the same optimization procedure adjust a a the instrument settings and set the scatter gate on a the population of inte
101. ion resolution in each detector and creates Baseline Calculations Report see page 58 Q IMPORTANT Make sure to perform baseline calculations after any major troubleshooting or cytometer service 1 Log on to the Attune Cytometric Software 2 Click Baseline Calculations on the Main Menu Attune Acoustic Focusing Cytometer User Guide 99 3 Prepare Attune Performance Tracking Bead suspension see page 54 Check the beadlot number of the beads you are using The beadlot number is the first six digits printed on the label disregard the alpha numeric characters Ne Note Manual Valve must be set to Sip Tube to perform baseline calculations 5 Select the appropriate beadlot number from the dropdown menu and go to step 9 Expiration Date a 4449754 Lot 756080 If the lot number of the beads you are using is not available in the dropdown menu proceed to step 6 The expiration date of the beads is displayed in the field above the dropdown menu and Run Baseline Calculations button becomes active Expiration Date February 11 2013 4449754 Lot 756080 v Run Baseline Calculations 6 If you are using a new lot of Attune Performance Tracking Beads click Get New Lot The Open dialog box appears Look in Beadlots E ex EJ E 4449754 Lot 756080 csv File name pO Files oftype Beadlot Fies esv xz __ Cancel 7 Select the appropriate beadlot file cs
102. ions during data acquisition may result in prompts and warning messages If you lower and raise the tube lifter during a cycle prior to the last sample draw in a run requiring multiple sample draws the following message will appear Attune Cytometric Software A Please check your sample Press OK to continue or Cancel and lower the tube lifter to rinse xan a Clicking Cancel initiates the Rinse operation once the tube lifter is lowered If you lower the tube lifter and leave it down during a cycle prior to the last sample draw in a run requiring multiple sample draws the following message will appear Attune Cytometric Software Please check your sample Press OK when sample is loaded and tube lifter is raised or press Cancel to abort the run Clicking Cancel aborts the run and automatically initiates Rinse If you lower the tube lifter in the last cycle of a run requiring multiple sample draws or during a single cycle run the following message will appear Status applied biosystems i The sample tube was lowered during the run Lower tube lifter to begin Rinse operation Lower tube lifter Cancel If the tube lifter is down Rinse operation automatically starts If the tube lifter is up lowering the tube lifter initiates the Rinse If the tube lifter is up Cancel is clicked and then Run is clicked the following message will appear Attune Cytometric Software PL Pleas
103. isplay of selected gates within the plot Back Gates This option allows you to display descendent gate data on a plot Only gates that are descendents or are at the same level within the hierarchy of the gates are available for display Back Gates can only be displayed on Dot Plots New Gates This option allows you to create new gates Rectangular Oval Polygon Quadrant Histogram and Bi Marker without inserting them from Gating Tools grouping within the Insert tab of the Ribbon menu Set Populations This option allows you to choose to limit the data displayed on a plot to a given gate It makes the plot a daughter of the upstream gate Create Daughter Plots You can also create plots as daughters from other plots by right clicking on a gate of interest selecting Create Daughter Plot and choosing the type of plot to display Dot Plot Density Plot or Histogram Scatter Test Sample Use Compensation Statistics i R Show Gates b l Back Gates f Delete New Gate P Hide Set Population 2 l Create Daughter Plot a Export to FCS File _ Customize Attune Acoustic Focusing Cytometer User Guide Working with Statistics Types of Statistics Global Statistics Statistics fall into two categories Global and Local statistics e Global Statistics show statistics for the full gating hierarchy from All Events down e Local Statistics show statistics f
104. ivileges to set manual Laser Delay Area Scaling Factor and to disable the bubble sensor All users have access to LinLog Linearization Parameter found under Advanced Options Instrument Settings Experiment 10 26 2010 02 56 PM Instrument Settings You can use the following options to update instrument settings Parameters Threshold Voltage Custom Parameters Advanced Options Laser Dely SS _ Use following manual laser delay 440 Last performance test laser delay 308 Blue Laser Area Scaling Factor C Use following manual blue laser area scaling factor 0 05 Last performance test blue laser area scaling factor 1 24 Violet Laser Area Scaling Factor Use following manual violet laser area scaling factor oos Last performance test violet laser area scaling factor 1 17 Other Options an Instrument Settings Experiment 07 01 2011 09 34 PM Instrument Settings You can use the following options to update instrument settings Parameters Threshold I Voltage Custom Parameters Advanced Options Laser Delay Last performance test laser delay N A Red Laser Area Scaling Factor Use following manual Red laser area scaling factor t a Last performance test Red laser area scaling factor N A Blue Laser Area Scaling Factor JUse following manual Blue laser area scaling factor a Last performance test Blue laser area scaling factor N A Other Options
105. lat panel monitor e Visual display of system status on instrument e Instrument startup lt 15 minutes and automated shutdown e User changeable keyed filters e Fully automated and manual compensation modes e Audible noise lt 50 dBA at 0 5m e Software allows data acquisition and analysis width height and area measurements and controls the instrument e Output file format FCS 3 0 e Live gating with automatic saving e Operator and administrator log in Attune Acoustic Focusing Cytometer User Guide 69 Operation Principles and Technical Overview Sample Loading Acoustic Focusing Sample Interrogation 70 The Attune Acoustic Focusing Cytometer is a benchtop cytometer that uses acoustic pressure to confine the injected particles to a tight central line along the axis of the capillary as the sample passes through the optical cell for interrogation AB Stems Attune acoustic f ic focusing cytometer This section explains how the Attune Acoustic Focusing Cytometer measures scattered light and fluorescence as cells pass through the laser beam The sample is loaded into the Attune Acoustic Focusing Cytometer via the sample injection tube which automatically delivers the sample to the flow cell after the tube lifter is engaged and the user defines collection criteria The sample and the focusing fluid are pushed through a capillary assembly where it is intercepted by the laser beam for interrogation The capillary
106. le All cytometers are governed by Poisson statistics which predict the probability of Concentration a given number of cells or particles being intercepted by the interrogating laser beam While increasing the sample concentration results in a higher sample throughput it also increases the probability of a coincident event defined as more than one cell present in the interrogating laser beam The Attune Acoustic Focusing Cytometer can maintain its maximum particle analysis rate over a large range of initial sample concentrations without the need to concentrate using centrifugation or filtration The ability to analyze dilute samples has the added benefits of reduced background fluorescence from free fluorophores in the sample and capability to analyze very small initial samples sizes Effects of Acoustic Acoustic focusing differs fundamentally from ultrasonic lysis of cells and is Focusing on Cell generally gentler on cells than the forces occurring in hydrodynamic focusing Viability Ultrasonic lysis of cells relies on cavitation produced at sub megahertz frequencies where tiny gas bubbles form and collapse with immense local shear and heating in the solution containing the sample In contrast the acoustic focusing capillary of the Attune Acoustic Focusing Cytometer operates at a frequency well above 1 MHz where the possibility of cavitation is greatly reduced Further acoustic cytometry is performed with relatively low energy levels at ve
107. leshooting Observation Possible Causes Recommended Solutions No events are displayed when in Run mode Threshold is not set correctly Ensure that threshold is set correctly and or for the right parameter on the instrument configuration panel Threshold level is too high Lower the threshold using the slider bars on the instrument configuration panel PMT voltages set too high or too low for parameter Optimize PMT setting by adjusting voltages up or down Sample may be too dilute Increase the sample flow rate No sample in tube Add sample or install new sample tube Clog in Sample Injection Port SIP e Run Unclog function e Run4mL of Attune wash solution using standard sensitivity with 500 uL minute flow rate Incorrect filter in detection channel Verify standard optical configuration Gating issue Verify that the plots are set to all events and or the gate logic is correct Laser is not functioning Verify laser function by re running the Performance Tracking function If the problem persists contact Technical Support Cells have been lysed e Ensure that the cells have not been lysed or broken up e Ensure that your sample contains cells Population is off scale e Adjust the axis to view the population e If events are detected in the counter ensure that the axis is set correctly Sample is not aspirated or only partially aspirated
108. lows you to save FCS Files Workspace and Instrument Settings and to print the Workspace objects It also lets you open the Options window access the Main Menu Screen and logout of the software Attune Acoustic Focusing Cytometer User Guide Instrument Settings Parameters Tab Instrument Settings allows you to specify Acquisition Parameters Threshold Voltage Custom Parameters and Advanced Options only available to users with administrative privileges on the Attune Acoustic Focusing Cytometer To access click Instrument Settings on the View Tab or double click the Instrument Settings icon within the experiment CO BR bB FS Instrument Sawe As File Settings Template Experiment Parameters tab allows you to select parameters for acquisition and name parameters at a global level Instrument Settings Experiment 10 26 2010 02 56 PM Instrument Settings Experiment 07 01 2011 09 34 PM instrument Settings Instrument Settings You can use the following options to update instrument settings You can use the following options to update instrument settings Parameters Threshold Voltage Custom Parameters Advanced Options Parameters Threshold Voltage Custom Parameters Advanced Options i Include Event Count Include Time Include Time Enabled Name i i Enabled Name Area Height Width Mf Fsc Zl rsc K K o KI ks E KI ks El HA 8 ks K K K K K K K K K K E E
109. ment and then collecting the rest under a second experiment The optical filters and mirrors are housed in optical holders which are located in the optics compartment To clean optical filters and lenses follow the instructions elow CAUTION LASER HAZARD Follow the precautions outlined in Laser Safety on page 158 while changing optical filters and mirrors The optical filters and mirrors are housed in optical holders which are located in the optics compartment To clean optical filters and lenses 1 Lift the top cover of the cytometer The photograph here shows the Blue Violet configuration The location of the optical filters and mirrors are the same in the Blue Red configuration Optical filters and mirrors Remove the optical holder containing the appropriate filter or mirror Attune Acoustic Focusing Cytometer User Guide 47 555 DLP 3 Gently remove any dust from the surface of the filter or mirror with a blower bulb blower or compressed gas or a soft brush 4 If necessary gently clean the surface of the filter or mirror using a clean lens cloth and lens cleaning fluid or methanol 5 Return the optical holder back to its slot and close the cover of the cytometer oN E A x b N Note The optical holders fit into the slots only one way Fluidics To ensure reliability of the instrument we recommend a monthly decontamination Decontamination of the fl
110. mercial applications of any kind including without limitation quality control and commercial services such as reporting the results of purchaser s activities for a fee or other form of consideration For information on obtaining additional rights please contact outlicensingfalifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Part Number 4453328 Rev F Contents PO OUC UNIS GULI Cixi cect oine rr room 9 Fow 10 D bal Up OL E KA aa ka aaa pra N SEK R NEK Ke E ee we e ek RS K N Veke KK an edeb 6 Sale IM Orma O Nesans a aa aa 4 to Daly ROUNE sun yy Aa yA xain a l NAYA aA Y N AW E E Xer Ki aS al kade k a k 9 WN OPK TOW od ccco e r r yryeIDKKO Kbbbbgmuoommrrrrmmn 9 Perre r ou N MD ggg9g99oa 2zm o 10 SR Pasa sales coupes oad nace atic ggo_ _ _ _ _ r ow gt gt 5 2a 11 NUL ECO WY Tatas ess AR E e de eye O AS e N E EI e S Avedon ne sy uae as 13 2a RUDNIN SAM PLCS l kiy a HAO ee An e AR SER NN DKRN e TR NO N n den Kine D r Kake kan Eba ek we 15 UV OL K LOYEK yey Semaya re Sek ad aa a ean ehh e are Ses i ae d AA 15 Betor You TBC sun a SN ae aa ec alittle al HA ay a eal anek n 16 Creat an Exp lllll ll earn A kan nebe b par e k edeb n ebe W e en k ey n N
111. mple Parent Gate e Plot Type selects Histogram Dot Plot or Density Plot e Chart Title allows you to enter the chart title e Style defines the font size and background e Border specifies the width and color of the border e Include Name of allows you to include Experiment Specimen and Sample and Parent Gate names in the chart title ys Note You can define default styles for all Workspace items using the Options dialog box Default styles are saved for each user 116 Attune Acoustic Focusing Cytometer User Guide In the Include Name of section check the desired box es for Experiment Specimen and Sample to include those names in the title The plot title is updated when you switch Samples For example if you checked all three the chart might look as follows Blue Laser New Experiment Specimen Sample a BL2 H re e Some of the options in the General Tab are specific to certain plot types Use Shading Allows you to shade the area below a Histogram Plot Resolution Allows you to determine the resolution of Histogram and Density Plots Density 100 1 Allows you to determine the zenn percentage of data points displayed on the selected Dot or Density Plot e To display an overlay plot drag and test sample test sample unstained cells drop any Sample into any existing plot except itself The legend is displayed above the plot with the names of each Sample in the co
112. nd add it to the water tank Store the Attune Shutdown Solution at 15 30 C The Attune Performance Tracking Beads are designed for use with Attune Cytometric Software to automatically characterize track and report performance measurements of the Attune Acoustic Focusing Cytometer The beads are used to define a baseline and conduct daily performance tracking of the cytometer Each vial contains enough beads for 50 measurements Store the Attune Performance Tracking Beads at 2 8 C Note Prior to use you need to download the appropriate lot specific file into the Attune Cytometric Software for each new lot of Attune Performance Tracking Beads Attune Acoustic Focusing Cytometer User Guide Attune Acoustic Focusing Cytometer User Guide 91 Appendix D Attune Cytometric Software Description About the Software Software Layout 92 The functions of the Attune Acoustic Focusing Cytometer are controlled by Attune Cytometric Software By default the software is installed in the C Program Files Applied Biosystems folder and the Attune Cytometric Software icon i e shortcut is placed on the computer desktop and under Start gt All Programs gt Attune Cytometric Software Note If you need to reinstall the Attune Cytometric Software for recovery purposes you must have Administrator access The Attune Cytometric Software is a flexible data acquisition and analysis tool tha
113. nd the wash and shutdown solution tanks have 500 mL capacity each Do not overfill the fluid tanks Screw the lid back on without over tightening it Replace the tank by sliding it into the appropriate slot and plugging the tubing back into instrument Q IMPORTANT For all fluid tanks always connect the tubing connector first Connecting the sensor cable while leaving the tubing disconnected may result in increased back pressure and damage to the fluidics system and introduction of air into the system Attune Acoustic Focusing Cytometer User Guide 11 Qa Note The fluid levels are monitored via floating sensors in all fluidics tanks When the fluid level is low or the waste bottle is full the software displays the appropriate warning message The blue LED on the affected fluid tank will also pulse To resume the run follow the displayed instructions and click OK Power On the 1 Power on the Attune Acoustic Focusing Cytometer by pressing the switch Cytometer and b located on the upper right corner above the fluidics door Computer 2 Power on the computer and monitor Wait for the computer to boot 3 Log in to Windows The default credentials are e User name INSTR ADMIN e Password INSTR ADMIN Y gt Note You can power on the instrument and the computer in any order Launch the 1 Launch the Attune Cytometric Software Software and Run If the daily Startup function has not been pe
114. ne Acoustic Focusing Cytometer s product page and import the lot specific data file The Beadlot data file has csv format 2 Save the Beadlot data file in C Programs Applied Biosystems Attune Performance Tracking Beadlots Alternatively once you have downloaded the beadlot file click Get New Lot to navigate to and select the beadlot file It will automatically populate the drop down list and be copied to the Beadlots folder Attune Acoustic Focusing Cytometer User Guide Baseline Calculations How It Works Performing Baseline Calculations The Baseline Calculations workflow of the Attune Cytometric Software uses Attune Performance Tracking Beads to define the cytometer s initial baseline status During this process the median fluorescence intensity of each bead and the HPCV percent half peak coefficient of variation are automatically measured in all fluorescence detectors Software algorithms use this information to determine cytometer settings and provide target values for subsequent application specific settings The software guides you through this process to measure the following values for each fluorescent bead using assigned MESF molecule of equivalent soluble fluorophore values e Relative detector quantum efficiency rQ e Relative background level rB e Linear regression e Laser delay setting The Attune Cytometric Software then automatically adjusts the PMT voltages to maximize populat
115. nge the most popular options in Attune Cytometric Software Administrator User Management Default Files and Folders Styles Default Workspace C Program Files Applied Biosystems Attune Users Admin Templates Def Resources Default Instrument Settings C Program Files Applied Biosystems Attune Users Admin Templates Def Templates Folder C Program Files Applied Biosystems Attune Users Admin Templates O Browse Other Options Default Sample Name Default Specimen Name E Show sample s recorded date time in experiment explorer 2 Click General to designate the default Workspace instrument settings and templates folder as well as changing the Default Sample and Specimen names 136 Attune Acoustic Focusing Cytometer User Guide Modes of Operation The functions of the Attune Acoustic Focusing Cytometer are controlled by Attune Cytometric Software The system has three cycles e Startup e Collect Run Rinse e Shutdown Startup Cycle The Startup cycle is performed at the beginning of each day to ensure that all fluidic lines are clean and that the fluidic lines and the system s two pumps are filled with fresh focusing fluid Collect Run Rinse The Collect Run Rinse cycle is performed during normal operation with data Cycle collection In the Run portion of the cycle there are two modes for data collection standard and high sensitivity each with several s
116. ning properly and the circuit breaker is not tripped Faulty power supply Contact Technical Support No fluorescent signal Incorrect parameter is selected Ensure that the correct parameter is selected Incorrect filter is installed Verify that appropriate filters are installed for each detection channel Laser is not functioning Run Performance Test to verify laser function If laser is not functioning properly contact Technical Support Incorrect fluorochrome Verify that the reagent excitation emission spectra match the collection filter set Reagent has degraded Restain your sample with fresh reagents Laser delay is incorrect Run Performance Test Event rate is too high Air bubble in focusing fluid filter or flow cell Run De bubble function Threshold is set too low e Increase the threshold level to reduce noise e Ensure that the threshold parameter is not set to Ignore PMT voltage for threshold is too high Lower the PMT voltage for threshold parameters Sample may be too concentrated e Lower the sample flow rate e Dilute the sample Sample flow rate is too high Lower the sample flow rate and or change the transit time to standard sensitivity mode Bacterial contamination Ensure that the sample is not contaminated Attune Acoustic Focusing Cytometer User Guide 141 Observation Possible Causes Recomm
117. nt plots Manage your back gates below Available Gates _ Back Gates EE gt BETTER O R3 e To apply a gate to the selected plot select the desired gate and click Add e To remove a gate from the selected plot select the desired gate and click Remove e To change the order of display for the back gates re arrange the back gates using the Move Up or Move Down buttons In the plot back gates at the top of the list are displayed on top of back gates down the list 120 Attune Acoustic Focusing Cytometer User Guide Gating Tools Gate Types Gating Tools allow you to isolate a region in a selected plot for analysis Hook Wo i Rectangular Oval Polygon Quadrant Denved Histogram Bi Wlarker Gate Gate Gate Gate Gate Gate Gating Tools A Rectangular Gate can be inserted into a Dot Plot or Density Plot An Oval Gate can be inserted into a Dot Plot or Density Plot A Polygon Gate can be inserted into a Dot Plot or Density Plot To use a polygon gate click on the plot to insert a handle Continue to insert handles to draw the gate around the population of interest To complete the process click on the original handle or double click when inserting the last handle You can stretch the polygon to any shape by clicking on a handle and dragging it to the desired location A Quadrant Gate can be inserted into a Dot Plot or Density Plot Quadrant Gates have an additional fea
118. number of the performance tracking beads you are using is identical to the beadlot number used in current baseline ys Note If you are using a new lot of Attune Performance Tracking Beads download the appropriate lot information see Using a New Lot of Beads on page 54 and perform new baseline calculations see page 55 6 Click Run Performance Test to initiate the automated Ran PETOA ETER Performance Test f The Workspace displays the acquisition plots and events appear in the plots as the Attune Cytometric Software proceeds with the performance test ETTOTGTTIT TT Attune Cytometric Software v2 1 Admin Ss am BS pled SB biosystems Scatter Blue Laser Violet Laser FSC H SSC H BL1 H ERT EE g FB a AS n 0 0 00 05 10 15 20 10 10 104 10 10 10 104 10 00 O05 10 15 20 00 O05 10 15 20 10 10 104 10 FSC 10 6 BLI VLI FSC H 10 6 SSC H 10 6 BLI H BL2 H BL3 H VL1 H VL2 H 2 104 10 VL1 H VL2 H 10 10 iy ral OD Qe EE 5 17 2012 The progress bar provides an ongoing update during the run and displays the steps of the performance test Adjusting laser delay on BLI Y Note The performance test takes approximately 15 minutes to complete At the end of the run the following message appears on th
119. o or more individual gates SCATTER R1 OR R2 8 7 pirsan rm 8 7 7 6 6 55 5 Z R1 OR R2 Z4 T T 3 3 2 2 1 1 eve 0 0 R1 OR R2 0o 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 FSC A 10 6 FSC A 10 6 e NOT gates include all events found outside the gate NOT SSC A 10 6 oO BA N O O a N co SSC A 10 6 O N A A OQ N 6 g 012 3 4 5 6 7 8 FSC A 10 6 FSC A 10 6 Derived Gates can also be used to create other derived gates Derived Gate Derived Gate Select gates and operator for a derived gate Gate Definition A RADDDDSa _ _a_ _ NOT w Gate Color a ae Attune Acoustic Focusing Cytometer User Guide 123 R1 OR R2 NOT R1 OR R2 NOT R1 OR R2 _____ _ gt SSC A 10 6 oO A N wo oO a N Co SSC A 10 6 O N Fk OA DN R1 OR R2 0 1 2 3 4 5 6 Ff 8 0 2 3 4567 8 FSC A 10 6 FSC A 10 6 hY Note The color of the derived gate cannot be changed once it is created You must select the color during the initial creation j A Histogram Gate can only be inserted into a Histogram Plot Histogram Gate LL A Bi Marker Gate can only be inserted into a Histogram Plot Bi Marker bate Customizing Gates Right clicking on a gate allows you to customize the gate name opacity fill color population color and border width and color Customize Customize You can use the following options to customize workspace item
120. ollow this cleaning procedure 48 Attune Acoustic Focusing Cytometer User Guide Periodic Maintenance Replacing the Visually inspect the syringe pumps daily for leaks Replace the syringes if you observe leaks from the syringe assembly and or there is erratic or no fluid draws up from the fluidics tanks or the sample injection port To replace the syringes Syringes 1 Execute the Shutdown function with 10 bleach The plunger drive will be lowered and the cytometer will power off automatically see page 13 2 Open the Syringe Pump door located on the left side of the cytometer see Syringe Pump Compartment on page 87 3 Remove the plunger lock screw and unscrew the syringe from the valve by rotating it counter clockwise 4 To install a new syringe pull the plunger down and align the syringe with the syringe port of the valve and rotate clockwise until the syringe end cap seal hits the bottom of the valve After bottoming out rotate clockwise 1 4 turn to ensure complete seal without over tightening Q IMPORTANT Failure to properly align the syringe when engaging the valve may lead to cross threading No tools should be used for tightening and securing the syringe to the valve Over tightening the syringe beyond the above recommendation could result in damage to the syringe and the valve 5 Align the hole in the plunger with the hole in the plunger holder assembly insert the plunger lock screw and tighten
121. omultiplier tube PMT voltage setting Detectors which measure the output from the 638 nm laser red on the blue red configuration Standard deviation Sample injection port Side scatter Detectors which measure the output from the 405 nm laser violet on the blue violet configuration Attune Acoustic Focusing Cytometer User Guide 5 How to Obtain Support User Documentation For the latest services and support information for all locations go to www lifetechnologies com support At the Technical Resources page you can e Search through frequently asked questions FAQs e Submit a question directly to Technical Support e Order Life Technologies user documents Safety Data Sheets SDSs certificates of analysis and other related documents e Download PDF documents e Obtain information about customer training e Download software updates and patches In addition the Support page provides access to worldwide telephone and fax numbers to contact Life Technologies Technical Support and Sales facilities When contacting customer support for instrument troubleshooting provide the instrument model and the instrument serial number Convey to the technical support any error messages that were displayed on your instrument and any troubleshooting that you have already performed see Appendix E Troubleshooting The guides listed below are shipped with the Applied Biosystems Attune Acoustic Focusing Cytometer Gui
122. or a particular branch of the gating hierarchy and are plot specific That is only gate data relevant to that plot parents and children are displayed Global statistics display the entire gating hierarchy for all plots Experiment Name 4 Peak Performance Testing Specimen Name Specimen Sample Name 041210 AM Name gz Al events E O Singlets H a Jeu rsc _ ssc H 5 2 H E 5 3 H vL v 2 4 vL3 H S L Singlets a a su a C FSc A E ssc a _ B 2 A _ 513 4 vi2 4 vL 3 A E v a Event Count 11 790 10 436 2 519 2 519 2 518 2 516 2 520 2 508 2 506 2 496 2 496 2 496 2 499 2 499 2 481 2 480 2 500 Attune Acoustic Focusing Cytometer User Guide Median X 856 640 855 691 2 147 267 852 993 816 110 2 299 107 2 231 868 2 454 312 2 325 277 2 661 919 732 734 2 127 868 731 005 741 036 2 273 025 2 207 381 2 128 075 2 436 205 2 248 821 CV X 32 54 2 38 1 77 3 10 8 14 1 33 1 76 1 78 1 58 2 36 3 37 2 19 3 50 7 86 2 12 2 06 1 90 2 48 2 36 SSC A 10 6 Count ay SC H 10 6 wn e amp Count 041310 AM Singlets A 00 05 10 15 20 FSC A 10 6 41310 AM 1000 a A WA o 104 BL1 A 310 AM a00 NE A 400 200 of 1 ill VL1 A 041310 AM 2 0 1 5 4 1 04 o54 0 0 Singiote Hi r r 00 05 10 15 20 FSC H 10 6 041310 AM 800 BL1 H 600 400 10 BL1 H
123. or move the instrument after it has been installed do not attempt to lift or move the instrument without the assistance of others the use of appropriate moving equipment and proper lifting techniques Improper lifting can cause painful and permanent back injury Depending on the weight moving or lifting an instrument may require two or more persons 1 WARNING Do not attempt to lift or move the computer or the monitor without the assistance of others Depending on the weight of the computer and or the monitor moving them may require two or more people Things to consider before lifting the computer and or the monitor Make sure that you have a secure comfortable grip on the computer or the monitor when lifting Make sure that the path from where the object is to where it is being moved is clear of obstructions Do not lift an object and twist your torso at the same time Keep your spine in a good neutral position while lifting with your legs Participants should coordinate lift and move intentions with each other before lifting and carrying Instead of lifting the object from the packing box carefully tilt the box on its side and hold it stationary while someone slides the contents out of the box Ensure that anyone who operates the instrument has Received instructions in both general safety practices for laboratories and specific safety practices for the instrument Read and understood all applicable Safety Data Sh
124. ortcuts Cut Copy Paste You can invoke Cut Copy Paste commands using context menus the ribbon and Functions keyboard shortcuts for all Workspace objects For example right click on a Dot Plot to display the context menu The context menu that is displayed depends on where you right click on the plot Blue Laser Sample Red Laser Sample 200 J 6 j Time 10 _ FSC A 10 150 SSCA p BL1 A 10 BL2 A F z BL3 A 10 E 100 BL4 A j RL1 A 10 RL2 A j Cut Ctrl X FSC H sot Copy Ctrl C 50 sscH Delete Del BL1 H BL2 H 0 BL3 H BL4 H 5 6 7 8 RL1 H RL2 H FSC W Back Gates gt SSC W New Gate gt BL1 W 10 rer lala Duplicate 4 Save As Statistics 10 6 Set Population gt V BL2 W A Customize BL3 W BL4 W RL1 W RL2 W Cut Ctrl X Copy Ctrl C Delete Del Duplicate Save As Scale Customize e You can paste objects that are copied into the Workspace or into a Microsoft Windows application that supports pasting e You can drag the Workspace objects to a new location while holding down the CTRL key The selected item is duplicated when you release the mouse button e You can press the Esc key to cancel regular or copy drag This still preserves the selection and the canceled drag is not considered as an undo action You will still be able to undo an action previous to the aborted drag if any Attune Acoustic Focusing Cytometer User Guide 113
125. played by entering the minimum and maximum range for the chart N Note You can right click directly on the plot axis to change the parameter and or the scale 118 Attune Acoustic Focusing Cytometer User Guide Samples Tab You can use the Samples tab to turn samples on and off set the color for each overlay remove samples and change the way the samples are displayed Customize Customize You can use the following options to customize workspace items General X Axis Y Axis Samples Gates Back Gates You can drag and drop any sample from the Explorer tree to the workspace area to display the sample side by side You can also drag and drop a sample from the Explorer tree over an existing Dot or Histogram plot to display it as an overlay Options Display Mode Display active sample and selected samples below only Available Samples Enabled Experiment Specimen Sample Experiment Specimen Sample 2 Experiment Specimen Sample 3 In the Samples tab you can select one of the following display modes 1 Display active tube only 2 Display selected tubes below only 3 Display active tube and selected tubes below only To display a Sample as an overlay to the active Sample check the Enabled box for the Sample To set the color for Sample click the Set Color square to display the Set Color dialog box and then select the desired color for the sample To remove a sample from the list of overl
126. press Delete on the keyboard e To resize a window or panel click on the edge of the window and drag the edge to resize e Workspace windows are by default docked around the edges of the main window To move a window e g Experiment Explorer click on the title bar and drag it to the desired location e Right click on the title bar of any window to EET P r ail My Ex Floati show docking options Note that floating ku rs oan 1 a Ne ockable windows are not docked you can move them u Auto Hide around but cannot dock them to any edge a rn Ej ee e To re dock a window left click on the title bar and drag the window into the desired area on the docking map that appears on lh the work area Ca Attune Cytometric Software v2 1 Admin oo Te En a Home Insert ompens n View Piate Qi S iee Collection Panel n __ e f e g 4 e he Expenments Sample 1 Double click to edit text Click and drag title bar to move a mee nia 0 events en Global Instrument Settings 4 Compensation Fa Blue Laser Sample Violet Laser Sample IM Unstained Contro Stop recording when Ji eL Any of these conditions are met 1 Jk 8L2 v 10 000 Events on Ali Events d j Ti BL3 5 Min 0 Sec at jk vu 4000 pl r 10 ji v 2 Record continuously z j E 2 3 Ji ve v i A 12 26 2011 06 34 PM i 1 1 we 4 12 27 2011 0553 AM gt _ fan EQ Global Workspace sa nae Se Ta amp Global Instrument Settings Ri
127. que FCS file Note If you set the collection criteria parameter to Record continuously then the data will be recorded until either the Acquisition Volume is exhausted or the maximum number of events allowed is reached If you select more than 10 parameters the event maximum is 500 000 If you select 10 or fewer parameters the event maximum is 20 000 000 Attune Acoustic Focusing Cytometer User Guide 37 Appending Data After recording data you can append the data file with additional data If you wish to append a data file for a Sample you must not modify the Instrument Settings or collection rate or change the Sample e g go to the next Sample However you can execute a Rinse function prior to appending data To append data 1 Click Run button and then click Record A dialog box opens and prompts you to select Append Overwrite or Cancel Attune Cytometric Software Specimen unstained cells fcs has existing data How should newly collected data be saved 2 Click Append if you wish to add the new data to the existing FCS ye Note When appending data the stop criteria is based on the individual record cycle and not the total resulting FCS file For example a file containing 10 000 events appended with a run that has stop criteria of 20 000 events will result in a file with 30 000 total events 38 Attune Acoustic Focusing Cytometer User Guide Warning Messages Various user act
128. r 14 2011 9 23 AM F e DEN Cd 12 14 2011 09 27 AM lC 12 26 2011 06 34 PM lC 12 27 2011 05 53 AM lC 12 27 2011 05 53 AM 1 lC 05 17 2012 02 30 PM New Experiment No template No template No template No template defined defined defined defined Click here to select one Click here to select one Click here to select one Click here to select one Blank Tube Experiment Blank Plate Experiment None None None Non No template No template No template No template No template defined defined defined defined defined Click here to select one Click here to select one Click here to select one Click here to select one Click here to select one None None None None None Browse Templates CaS Functions A e m amp om 3am 5 7 2012 _ Note Results of cytometer performance tracking tests are available for all users under the Reports option on the Quality Control menu but the Baseline Calculations and Performance Test options are available only to authorized users Attune Acoustic Focusing Cytometer User Guide 93 Attune Performance Tracking Beads Preparing Attune Performance Tracking Bead Suspension Using a New Lot of Beads 54 The Attune Performance Tracking Beads are used to define a baseline for cytometer performance and conduct daily performance measurements of the cytometer They are stained with a combination of fluorophores that can be excited by the lasers used in the Attune Acoustic Fo
129. red Other Workspaces and Instrument Settings for the experiment can be selected in this at this point Attune Acoustic Focusing Cytometer User Guide Experiment Experiment You can use the following options to add or update an experiment Experiment Name 102720100502 PM Description Time Created 10 27 2010 5 02 PM Time Modified Workspace C Program Files Applied Biosystems v1 1 20 Attune Users Admin Tem C Program Files Applied Biosystems v1 1 20 Attune Users Admin Temy Samples Create Specimen s for this Experiment Create 1 Sample s per Specimen Note For information on setting the default Workspace and Instrument Settings see General Options on page 136 Duplicating a 3 From Experiment Explorer right click on Saved Experiment the Experiment you want to duplicate and choose Duplicate Experiment 4 Check the desired options in the Duplicate Experiment Options dialog box then click OK Workspace opens and displays the same objects as the duplicated experiment but without the data New Specimen Duplicate Expenment Delete Experiment Rename Experiment Export Experiment Export FCS File s Export Statistics Print Open Expenment Folder Experiment Properties oo li Duplicate Experiment Options x Duplicate Experiment Options Use the following options to select what to duplicate Compen
130. rest MN 7 4 For each compensation control sample i e d a Specimen fluorophore observe the corresponding I sample histogram to optimize the voltages 5 Perform the optimization procedure for all single stained controls 6 After you have optimized instrument settings for each single stained control IMPORTANT Once you click Record on any of the compensation controls within the compensation setup the Instrument Settings for all fluorescent channels will be grayed out and cannot be changed It is critical that you optimize voltages prior to recording any sample or any compensation controls iy LF i e Note The Scatter Gate defined in the Unstained control can be applied to all other compensation controls by right clicking the gate R1 and selecting Apply gate shape to all controls R1 Customize Note The Attune Cytometric Software automatically executes the Rinse function each time the tube lifter is pushed down to remove the sample from the sample injection port This ensures that the fluidics system of the instrument is flushed and any remaining sample is removed to minimize carryover Attune Acoustic Focusing Cytometer User Guide 25 Calculate Compensation Fluorophores emit light over a range of wavelengths Although optical filters limit the range of frequencies measured by a given detector when two or more fluorophores are used in an experiment there is often
131. rface by reading Appendix C Instrument Description and Appendix D Attune Cytometric Software Description before starting your experiments Attune Acoustic Focusing Cytometer User Guide 7 Before You Begin Required Solutions e Attune Focusing Fluid is a buffered azide free support carrier reagent for transporting particles through the capillary assembly It contains a preservative and detergent designed to minimize bubble formation e Attune Wash Solution is a ready to use solution for removing cellular debris and dyes from the fluidics system of the instrument e Attune Shutdown Solution is a 10X solution that prevents bubble formation in the fluidics system of the instrument Prepare a 1 10 dilution of the shutdown solution in deionized water and add it to the water tank e 10 bleach solution in deionized water decontaminates the fluidics lines Prepare this solution fresh daily and use during the shutdown procedure e Deionized water used for diluting Attune Shutdown Solution and bleach as well as for long term storage of the instrument Q IMPORTANT 10 Bleach is defined as a 1 in 10 dilution 1 part bleach to 9 parts water of 5 25 sodium hypochlorite in water This gives a final concentration of 0 5 sodium hypochlorite equivalent to 5000 ppm of available chlorine Q IMPORTANT Reagents may be stored at colder temperatures but running the instrument with cold reagents lt 15 C will affec
132. rformance Tracking Experiment Explorer Performance Performance Lracking advances to baseline calculations and pertormance tests Tracking screens It also provides the last completed performance test and the run status 94 Attune Acoustic Focusing Cytometer User Guide New Experiment New Experiment allows you to create a new experiment using a blank template or a user defined or preset template that is pre populated The panel displays 10 user defined experiment templates New Experiment _ Eg TEWE r T A iV j L No template Finale ils defined Fr o pm Click here to select one e 2 No template No template No template No template No template defined defined defined defined defined Click here to select one Click here to select one Click here to select one Click here to select one Click here to select one Experiment Experiment Explorer Browse Experiments lists the experiments of the logged in Explorer Panel operator The experiments are stored in individual folders Browse Experiments amp 12 14 2011 09 27 AM 12 26 2011 06 34 PM 12 27 2011 05 53 AM og Global Workspace en Global Instrument Settings a H Compensation M Unstained Control 4 lC 05 17 2012 02 30 PM Ld Global Workspace Global Instrument Settings 4 Compensation 4 Specimen W Sample Double clicking on any Sample within the experiment opens the Workspace Note The Experiment Explorer obj
133. rformed Startup displays in the Startup Function upper right corner If the daily Startup function has already been performed then Shut Down displays in the upper right corner 2 Either click Startup on the upper right corner of the Login screen then log into the software by entering your Username and Password Or if Shut Down displays in the upper right corner enter your username and password then click Login aa Note The default username and password for the software are admin and password respectively After logging in assign a password to the administrator account as soon as possible To create additional users see Software Administration on page 133 The Startup prompt screen appears and provides instructions to perform the Startup operation The Attune Cytometric Software automatically turns on the lasers and instrument systems initializes the pumps and primes the fluidics lines The status window displays the Startup operation being performed After the Startup function is completed and no system errors are encountered the Status bar displays the Ready icon 1 If any system errors are encountered during the Startup the status bar displays the Alarm icon oN Note A flashing green light on the instrument indicates that Startup is under way and a continual green light indicates that the instrument is ready 12 Attune Acoustic Focusing Cytometer User Guide IMPORTANT When yo
134. rresponding color When overlaying histograms ensure that the resolution is the same otherwise the graph may not look the same Repeat the process to have more than one overlay e Only Histograms and Dot Plots can have overlays e To display plots side by side drag and drop any Sample from Experiment Explorer into a blank area in the Workspace and set the plot to the desired type and parameters Attune Acoustic Focusing Cytometer User Guide 117 X and Y Axis Tabs Using the X Axis and Y Axis tabs you can change the parameter and scale type set the range and format the appearance of the X and Y Axis title bars respectively The X Axis and Y Axis tabs have the same options available Customize Customize You can use the following options to customize workspace items General X Axis Y Axis Samples Gates Back Gates Parameter BL3 A v Scale Title Type Log x Text BL3 A Range Automatic Style Manual Tahoma Minimum 100 B r u Av v Maximum 6300000 Border Width 0 amp e Use the Parameter drop down menu to select the Parameter parameter you want to display Scale e You can set the Scale type to Linear Log or ka Linlog Range 2 Automatic e If Range is set to Automatic the scale will Manual incorporate the entire range of the data limits Minimum e If Range is set to Manual you can specify the Maximum scale to be dis
135. rt PMT malfunction Contact Technical Support Incorrect performance tracking beadlot used Verify the lot number and download the correct lot information High APMT in all Incorrect bead sample is used Prepare a new bead sample channels Incorrect performance tracking Verify that the software has the appropriate beadlot used beadlot data installed and re run the Performance Test Bead sample removed before the Re run the Performance Test in its entirety Performance Test was completed using the same bead sample Bead sample too dilute to register Prepare a fresh bead sample according enough events following manufacturer s recommendations Clog or partial clog in the flow cell Run Unclog function Particle s stuck in the flow cell Run long wash cycle Bubbles in the system Run De bubble function APMT in all channels Low or no laser power Contact Technical Support for a single laser Wrong optical configuration for a single laser Check the optical configuration Laser is misaligned Contact Technical Support Bubbles in the system Contact Technical Support High HPCV ina single channel for a single laser line High HPCYV in all channels for a single laser line High HPCV in two channels for a single laser line 144 Dirty emission filter Inspect and clean filter Incorrect emission filter Check the optical configuration PMT malfunction
136. rts are inside Refer servicing to Life Technologies qualified service personnel AVERTISSEMENT Pour viter les risques d lectrocution ne pas retirer les capots dont l ouverture n cessite l utilisation d outils L instrument ne contient aucune pi ce r parable par l utilisateur Toute intervention doit tre effectu e par le personnel de service qualifi venant de chez Life Technologies DANGER Class 3B visible and or invisible laser radiation present when open Avoid exposure to beam DANGER Rayonnement visible ou invisible d un faisceau laser de Classe 3B en cas d ouverture Evitez toute exposition au faisceau gt gt CAUTION Moving parts Crush pinch hazard ATTENTION Pieces en mouvement risque de pincement et ou d crasement 152 Attune Acoustic Focusing Cytometer User Guide General Instrument Safety Moving and Lifting the Instrument Moving and Lifting Stand alone Computers and Monitors Operating the Instrument Cleaning or Decontaminating the Instrument WARNING PHYSICAL INJURY HAZARD Use this product only as specified in this document Using this instrument in a manner not specified by Life Technologies may result in personal injury or damage to the instrument CAUTION PHYSICAL INJURY HAZARD The instrument is to be moved and positioned only by the personnel or vendor specified in the applicable site preparation guide If you decide to lift
137. rument meets European requirements for emission and immunity EMC Directive 2004 108 EC This instrument has been tested to and complies with standard EN 61326 Group 1 Class B Electrical Equipment for Measurement Control and Laboratory Use EMC Requirements This instrument has been tested to and complies with standard AS NZS 2064 Limits and Methods Measurement of Electromagnetic Disturbance Characteristics of Industrial Scientific and Medical ISM Radio frequency Equipment Attune Acoustic Focusing Cytometer User Guide 159 SDSs SDSs Chemical manufacturers supply current Safety Data Sheets SDSs with shipments of hazardous chemicals to new customers They also provide SDSs with the first shipment of a hazardous chemical to a customer after an SDS has been updated SDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new SDS packaged with a hazardous chemical be sure to replace the appropriate SDS in your files Obtaining SDSs You can obtain from Life Technologies the SDS for any chemical supplied by Applied Biosystems This service is free and available 24 hours a day To obtain SDSs 1 Goto www lifetechnologies com click Support and then select SDS 2 Inthe Keyword Search field enter the chemical name product name SDS part number or other information that appears in the SDS of interest Select the language of your choice
138. ry high sample flow rates and the design of the acoustically driven capillary spreads this energy over the entire length of the capillary significantly reducing the probability of cellular damage 74 Attune Acoustic Focusing Cytometer User Guide Optics Light Scatter Measuring Light Scatter Fluorescence When a cell or particle passes through a focused laser beam it refracts or scatters light in all directions e Forward scatter or low angle light scatter is the light that is scattered in the forward direction as laser light strikes the cell The magnitude of forward scatter is roughly proportional to the size of the cell or particle and this data can be used to quantify particle size e Side scatter is defined as the light that is scattered at larger angles Side scatter is indicative of the granularity and structural complexity inside the cell or particle Forward scatter Side scatter Forward scattered light is quantified by a detector that converts intensity into voltage In most cytometers a blocking bar called an obscuration bar is placed in front of the forward scatter detector The obscuration bar prevents intense laser light from reaching the detector As a cell crosses the laser light is scattered around the obscuration bar and is collected by the detector Side scattered light is focused through a lens system and is collected by a separate detector usually located 90 from the laser s path Fluore
139. s v Recover unused sample Bele lt 4 Collection controls Sample Dispensed O uL Acquisition Volume 300 uL 420 uL total draw volume 100 ut min t Collection mode EE lt 4 Sample rate t Collection P B instrument B Plate Setup B Heat Map al 4 Collection Panel tab You may switch between panels using the tabs ce gw ute LESPECUVE L114141c5 e Instrument Configuration YE Collection Panel The Collection Panel displays the cumulative count and event rate of all events that pass through the laser beam and register above the set threshold The progress bar provides an ongoing update of completion as defined by the user in stop criteria 00 ev sec 00 events 0 a Progress bar Attune Acoustic Focusing Cytometer User Guide 97 Using the Collection Panel you can set the collection criteria for continuous recording or you can prompt the instrument to stop recording when one or more of the following specified conditions is met e Number of events for the specified gate Stop recording when f 3 Any of these conditions are met e Elapsed time from the start of the Eray recording T Me e Volume of sample analyzed ul Record continuously Record continuously allows recording continuously until the full draw volume is exhausted Note When using stop criteria event time or volume the condition that is met first will take precedence
140. s General General Name R1 M Enabled Population C A 1076 Opacity 25 amp 7 Bw Border Width 2 Delete Hide Create Daughter Plot Export to FCS File Clicking on the gate name also allows you to edit the gate name without opening the customize window 124 Attune Acoustic Focusing Cytometer User Guide Export Gate to FCS Right clicking on a gate also allows you to export the gate data to a new FCS file ai e Exporting the gate data to a new FCS only exports the events that fall within the gate Whole Lysed Blood Scatter Whole Lysed Blood Scatter SSC A 1076 SSC A 1076 pi 2 3 4 5 6 2 3 4 5 6 FSC A 10 6 FSC A 10 6 Before Export After Export e If compensation is applied you will be prompted to store the compensation values in the FCS file The matrix is stored using the Spillover keyword Attune Cytometric Software FCS file contains no compensation matrix but the active experiment has a compensation matrix set Would you like to store this matrix in the FCS file xn axan acs Attune Acoustic Focusing Cytometer User Guide 125 Other Gate Options 126 Right clicking on a plot brings up a contextual menu that allows you to Pal rar Bn ee aes Si all a Ma Cut Cirl x Copy Ctrl c a Delete Del a a Duplicate q 5 GAVE AS T nt Th Show Gates This option allows you turn on or off the d
141. sation Settings Local Instrument Settings Local Workspaces The Experiment Explorer frame displays the duplicated experiment but the Sample icons indicate that they contain no data Attune Acoustic Focusing Cytometer User Guide 21 Optimize the Experiment Before You Begin 22 Before you can record data for a sample you need optimize your Experiment Experiment optimization involves fine tuning the PMT voltages compensation and threshold settings for each dye and sample used in the experiment to adjust the positions of populations of interest on scale for the scatter and fluorescence parameters Note We recommend that you optimize each individual experiment prior to collecting data If compensation is to be applied ensure that all Voltage settings except for FSC and SSC have been finalized for all samples prior to recording any data Compensation applied to samples that have voltages that are different from those used in the compensation setup may produce erroneous results If the experiment requires compensation prepare the necessary compensation controls You need an unstained or isotype labeled control and single stained controls for each fluorophore i e compensation beads or cells you are using for compensation Create an Experiment in the Experiment Explorer Create the necessary Specimens and Samples To create a Specimen right click the Experiment and select New Specimen To create a Sample righ
142. scence is the emission of light that occurs when an emitting particle such as a fluorophore labeled antibody absorbs light from another source such as the intercepting laser beam When the particle absorbs the intercepting light it is elevated to an excited electronic state and as it returns to its ground state the absorbed energy is radiated where most of it is emitted as light The emitted light is always a longer wavelength i e less energetic than the absorbed light The difference between the excitation and emission wavelengths is known as the Stokes shift Flow cytometry uses fluorescence detectors to identify different aspects of cells including functional assays and subset identification One of the most common ways to study cellular characteristics using flow cytometry involves the use of fluorescent molecules such as fluorophore labeled antibodies In these experiments a fluorescently labeled antibody is added to the cell sample The antibody then binds to a specific molecule on the cell surface or inside the cell When laser light of the right wavelength strikes the fluorophore a fluorescent signal is emitted and detected by the flow cytometer indicating a specific binding event Fluorescence data is collected in generally the same way as side scatter data Ina population of labeled cells some will be brighter than others As each cell crosses the path of the laser a fluorescence signal is generated The fluorescent light is Att
143. sing Cytometer This image shows the Blue Violet configuration The location of the Compartment l i i components is the same in the Blue Red configuration Excitation Beam shaping optics Storage for spare Collection Collection Recessed PMTs collection optics optics lens and amplifiers 88 Attune Acoustic Focusing Cytometer User Guide Status Indicator Lights The Status Indicator Lights above the sample injection port identify the status of the cytometer t 1 Startup pea Wash Trigger Ready Processing Running Da BE E e Instrument Cycle Green Red Blue Yellow Green Startup BLINK ON OFF ON OFF OFF Indicator light is ON for 10 seconds and then turns off Startup complete ON OFF OFF OFF OFF Idle ON OFF OFF OFF OFF Run ON OFF OFF OFF BLINK Indicator light blinks with cycle time proportional to event rate Run complete ON OFF OFF OFF OFF Wash Declog De bubble OFF OFF BLINK OFF OFF Rinse OFF BLINK OFF ON OFF OFF Indicator light blinks when the rinse cycle is initiated and turns off when it is okay to place the sample on the sample injection port Clog detected OFF BLINK OFF ON OFF Focusing fluid tank empty Waste tank full OFF BLINK OFF OFF OFF Wash tank empty Water tank empty Shutdown OFF OFF BLINK OFF OFF Error OFF ON OFF OFF OFF Attune Acoustic Focusing Cytometer User Guide 89 Instrument Reagents and Consumables Attune Focusing Fluid At
144. syringe pumps for any leakage Decontaminate any spills wiping the area with 10 bleach solution If you notice any leaks in the fluidics lines contact your service representative Fluidics Daily fluidics cleaning involves executing the De bubble Unclog and Wash Maintenance functions as needed e De bubble is a user initiated function for clearing bubbles in the fluidics lines of the cytometer e Unclog function is a user initiated back flush operation to remove clogs from the sample probe and flow cell e Wash is a user initiated system cleaning between sticky samples This function requires user supplied bleach or detergent 46 Attune Acoustic Focusing Cytometer User Guide Monthly Maintenance Computer Maintenance Cleaning Optical Filters and Mirrors Periodically maintaining the computer running the Attune Cytometric Software is an important component of a comprehensive maintenance strategy To preserve the integrity of your data observe the following precautions De fragment the hard drive of the computer monthly Back up your experiments on a regular basis to a secondary storage device When planning the experiments remember to delete parameters that you do not need i e only collect parameters in either area or height but not both unless you need both parameters for a certain application such as cell cycle If an experiment contains several samples consider collecting some of the samples under one experi
145. t click the Specimen and select New Sample Create a Workspace with the plots appropriate for your experiment To optimize instrument settings for compensation control samples open the compensation setup guide as described on page 26 and select the necessary parameters for compensation Compensation control samples are created within the Compensation al module in the Experiment Explorer See pages 23 25 for details about optimizing compensation controls If no compensation is necessary you can optimize the instrument settings within the Workspace of the Sample in the Experiment Explorer The procedure for optimizing samples is similar to that described for compensation control samples See pages 23 25 Adjust all voltages to put the population of interest on scale in all necessary channels It is recommended that voltages are set to maximize the signal to noise You can adjust the threshold and voltages using the Instrument Configuration tab on the Collection Panel Alternatively you can use Instrument Settings in the View tab which gives you the additional ability to rename parameters Attune Acoustic Focusing Cytometer User Guide Optimizing e We recommend that you optimize the Instrument Settings for compensation Instrument controls in the Compensation Workspace See Compensation Setup on page 26 e Optimize Instrument Settings for the unstained control and for each of the Settings for i single stained controls as follows
146. t the data quality Before you run the instrument ensure that all fluid temperatures are at least 15 C 10 Attune Acoustic Focusing Cytometer User Guide Startup Check Fluid and Waste Levels Fill the Fluid Tanks During Startup the Attune Acoustic Focusing Cytometer Warms the lasers to operating temperature Initializes the pumps Primes the instrument fluidics Informs the user of System Status Ready Attention Clog etc The Startup function ensures that all fluidic lines are clean the fluidic lines and the system s two pumps are filled with fresh focusing fluid and the lasers are warmed to operating temperature Check the levels in the fluid tanks see page 87 for the location of the fluidics compartment If empty fill the focusing fluid wash solution and shutdown solution tanks Q IMPORTANT Reagents may be stored at colder temperatures but running the instrument with cold reagents lt 15 C will affect the data quality Before you run the instrument ensure that all fluid temperatures are at least 15 C If full empty the waste tank 1 IMPORTANT The Attune Acoustic Focusing Cytometer must be powered off or in stand by mode before refilling the fluidics tanks Press the metal release buttons to free the tubing and sensor cables from the instrument and remove the tank Unscrew the lid and fill the tank with the appropriate solution The focusing fluid tank has 1 L capacity a
147. t uses a browser view to e Design and perform experiments e Define independent instrument settings and optimize data collection e Carry out instrument performance checks and track instrument performance e Acquire and record data e Manage and process recorded data The Attune Cytometric Software has three main screens e Login Used for logging into the software and running the Startup function e Main Menu Used for running and reviewing performance tracking tests creating new experiments and accessing the current and stored experiments of the operator It consists of three panels Performance Tracking New Experiments Experiment Explorer Browse Experiments e Attune Desktop Used for controlling the Attune Acoustic Focusing Cytometer to run samples generate data and analyze results Work Area Experiment Explorer Attune Acoustic Focusing Cytometer User Guide Login Screen The first screen that is displayed when you start the Attune Cytometric Software is the Login screen User Accounts The Attune Cytometric Software has two types of user accounts Administrator and Operator e Administrators have full access to the software and can perform a variety of tasks including adding users assigning data storage paths for user s folders and changing and resetting passwords e Operators can access and modify their own Workspace and experiments but cannot add or delete users After a successful lo
148. teria Met Violet SSC 700000 765829 00 1075 25 mV 0 10 N A N A N A Criteria Met Blue BLi 2000000 2156936 00 875 200mV 1 00 0 072 508 972 999 Criteria Met Blue BL2 2000000 2110261 00 1125 50 mv 0 80 0 177 98 599 1 000 Criteria Met Blue BL3 2000000 1960195 00 950 50 mV 4 30 0 037 125 194 1 000 Criteria Met Violet VL1 2000000 2155662 00 825 75mV 2 40 0 009 291 374 1 000 Criteria Met Violet VL2 2000000 1847469 00 1050 25mV 420 0 012 1514 161 981 Criteria Met Violet VL3 2000000 2112920 00 975 75mV 0 10 0 142 4 467 545 Criteria Met e The panel on left displays the performance test report history You can access previous performance test reports through this panel Current Baseline 5 17 2012 3 33 PM 756080 Lot Mo 756080 Expiration Date 05 20 2021 Current Baseline 4 Baseline 5 17 2012 3 33 PM 756080 wf Daily 5 17 2012 3 38 PM Previous Baselines Baseline 12 14 2011 9 23 AM 756080 vi This symbol indicates that the daily performance was successful The performance test results are shown as Criteria Met along with the date and time of the run A This symbol indicates that one or more parameters deviated slightly from the baseline values The performance test results are shown as borderline along with the date and time of the run This symbol indicates that the results of the performance test deviate significantly from the baseline values The perform
149. terials See SDSs page 160 e Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the SDS e Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS e Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS e Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal Attune Acoustic Focusing Cytometer User Guide Chemical Waste Safety Chemical Waste Hazard Chemical Waste Safety Guidelines Waste Disposal CAUTION HAZARDOUS WASTE Refer to Material Safety Data Sheets and local regulations for handling and disposal To minimize the hazards of chemical waste Read and understand the Safety Data Sheets SDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet
150. the events are displayed on the plots as the graphs are being populated e The software actively displays only 20 000 events during the run The statistics window displays only the total events once the recorded run has completed ys Note To refresh the data displayed on plots click F5 e Inthe Record mode the Attune Cytometric Software automatically creates an FCS file when Stop is clicked e The software creates a unique name for each FCS file so that an existing file is not overwritten Attune Acoustic Focusing Cytometer User Guide 30 36 If the Sample already has an FCS file the software displays a warning that the FCS file is going to be overwritten You can choose to append the existing FCS file or overwrite it see Appending Data page 38 Attune Cytometric Software Specimen unstained cells fcs has existing data How should newly collected data be saved Attune Acoustic Focusing Cytometer User Guide Preparing the Workspace Acquiring Data Recording Data Prepare your Workspace and customize your plots by selecting each individual plot one at a time Change the axis labels and other properties appropriate to your experiment Select the Sample of interest from the Experiment Explorer panel Double click Sample to activate it Workspace displays the default sample plots set up for the Experiment If desired you may delete or modify these plots Enter the collection criter
151. those used to acquire the compensation controls the user will be prompted with Attune Cytometric Software WARNING The voltages in the imported file are different from the experiment voltages The resulting compensation may not be appropriate for previously recorded data Updating the experiment voltages is recommended if additional samples are to be acquired Do you want to update the experiment voltages with the ones from the imported file Q IMPORTANT We recommend that you import Compensation Settings prior to recording any sample data so that the voltages used to acquire samples match those used to acquire the compensation controls If the voltages used to generate the Compensation Matrix do not match those used to acquire a sample applying compensation may produce undesirable results Attune Acoustic Focusing Cytometer User Guide 31 Saving the Compensation Matrix Resetting the Compensation Matrix 32 1 After creating a Compensation Matrix you can save it to a desired location by right clicking on the Compensation option in the Experiment Explorer and selecting Save Compensation Settings as 2 To use the saved Compensation Matrix in further experiments right click on the Compensation option in the Experiment Explorer and select Import Compensation Settings To reset the Compensation Matrix to the original calculated values open the matrix by clicking Compensation Matrix and then click the Reset
152. ths outside the selected interval which can be wide or narrow depending on the number of layers of the filter The bandwidth of the filter is simply the difference between the upper and lower cutoff wavelengths Common bandpass filter nomenclature is the peak emission bandwidth For example a filter that would detect Alexa Fluor 488 dye would be 530 30 which would allow wavelengths in the 515 545 nm range to pass 486nm BP 530 30 460 nm 520 nm Alexa Fluor 488 540 nm emission spectrum 700 nm BP 530 30 500 nm 600 nm Attune Acoustic Focusing Cytometer User Guide 77 Longpass Filter 78 Longpass filter LP is an optical interference or colored glass filter that attenuates shorter wavelengths and transmits i e passes longer wavelengths over the active range of the target spectrum ultraviolet visible or infrared Longpass filters which can have a very sharp slope referred to as edge filters are described by the cut on wavelength at 50 of peak transmission For example an LP 530 filter permits wavelengths longer than 530 nm to pass while reflecting or absorbing wavelengths shorter than 530 nm 700 nm 540 nm 460 nm LP 530 488 nm wy re Foe emission spectrum 520 nm 560 nm 600 nm Attune Acoustic Focusing Cytometer User Guide Shortpass Filter Dichroic Mirror Neutral Density Filter Shortpass filter SP is an optical interference or colored glass filter that attenuat
153. tton in the Quick Access toolbar Alternatively click Options under Global in the Home contextual tab Click User Management to select the user from the list displayed Click Properties to open the User dialog box and select the desired access type Administrator Operator Service from the Access Type dropdown menu Check the appropriate boxes to assign user privileges You can assign the following user privileges e Enable Viewing System Transactions e Enable Viewing User Transactions e Enable Running Baseline Calculations e Enable Running Performance Test Click Options La shortcut button in the Quick Access toolbar Alternatively click Options under Global in the Home contextual tab Click Styles to select the desired default styles for charts text objects statistics and gates Attune Acoustic Focusing Cytometer User Guide 135 General Options are specific to each user You can use this tab to set the default Workspace gws and Instrument Settings gis used when creating new experiments as well as the Templates folder General Options 1 To access General Options click Options Lit shortcut button in the Quick Access Bar in the upper left corner of the screen Alternatively click Options under Global in the Home tab _ n tas Optons Main Transaction Menu Log Global Options window opens Options Options You can use this to manage users and general application settings aa Lag Cha
154. tune Wash Solution Attune Shutdown Solution Attune Performance Tracking Beads 90 The following reagents are approved for use on this system Q IMPORTANT Reagents may be stored at colder temperatures but running the cytometer with cold reagents lt 15 C will affect the data quality Before you run the cytometer ensure that all reagent temperatures are at least 15 C The Attune Focusing Fluid is a buffered azide free support carrier reagent for transporting the samples through the Attune Acoustic Focusing Cytometer It contains a surfactant and a preservative For best results store the Attune Focusing Fluid at 15 30 C The Attune Wash Solution is a ready to use solution that has been formulated to remove cellular debris and dyes from the fluidics system of the Attune Acoustic Focusing Cytometer Store the Attune Wash Solution at 15 30 C The wash solution is stable on the cytometer for 30 days after the bottle has been opened The Attune Shutdown Solution is a 10X solution that prevents the formation of air bubbles and salt deposits in the fluidics system of the cytometer Trapped air bubbles in the fluidics lines can dislodge and result in inaccurate data as they pass through the flow cell and salt deposits can clog the lines The Attune Shutdown Solution is provided as a 10X solution that requires dilution before use Prepare a 1 10 dilution of the shutdown solution in deionized water a
155. ture that allows precise population demarcation These gates can be bended to fit a population better In the Default Quadrant Gate all lines are connected and form right angles as seen in the example below Lysed Whole Blood 10 10 End of Line Segment a 2 8 10 h ro z oO 5000 a N i y an Middle of Line 2500 a Segment 2900 Zot J 2500 11 4 BL1 CD3 FITC A Attune Acoustic Focusing Cytometer User Guide 121 122 In a Flexible Quadrant Gate the lines that demarcate the populations of interest do not form right angles as shown in the example below End of Line Segment Middle of Line Segment VL1 CD8 Pacblue A e Grabbing the middle of a line segment allows for the independent movement of each arm e Grabbing the end of a line segment allows you to hinge the arm about its attachment point Attune Acoustic Focusing Cytometer User Guide O A Derived Gate can be created as long as there is at least one gate u present in the Workspace You can create them with any or no plot Fa selected There are three types of Derived Gates e AND Intersected gates include all events that are shared between two or more individual gates SCATTER R3 AND R4 8 i 8 7 7 6 6 3 5 55 hand a R3 AND R4 lt lt 3 23 2 2 1 i yii 0 0 gt an R3 AND R4 0123 45 67 8 FSC A 10 6 FSC A 1076 e OR Joined gates include all events that are found within tw
156. u power on the instrument always allow at least Q 10 minutes for the lasers to reach operating temperature before you run samples Shutdown The Shutdown function of the Attune Cytometric Software facilitates the automated shutdown of the instrument The function ensures that all sample fluid and dyes have been removed from the system the fluidics lines and the two pumps have been decontaminated and filled with Attune Shutdown Solution to prevent the formation of salt crystals The automated shutdown procedure can take up to 30 minutes Q IMPORTANT Perform the following shutdown procedures at least once a day even if the instrument is intended for continuous use Proper cleaning of the instrument ensures its consistent and accurate operation CAUTION BIOHAZARD Cytometer hardware may be contaminated by biohazardous material Using fresh 10 bleach solution in deionized water is the only procedure we recommend for decontaminating the cytometer Q IMPORTANT 10 Bleach is defined as a 1 in 10 dilution 1 part bleach to 9 parts water of 5 25 sodium hypochlorite in water This gives a final concentration of 0 5 sodium hypochlorite equivalent to 5000 ppm of available chlorine Check Fluid and 1 Check the levels in the fluid tanks see page 87 for the location of the fluidics Waste Levels compartment 2 Ensure that the wash solution tank is at least half full If empty fill the tank with Attune Wash Sol
157. ugust 13 2005 Symbol Description Do not dispose of this product as unsorted municipal waste Follow local municipal waste ordinances for proper disposal provisions to z reduce the environmental impact of waste electrical and electronic lt equipment WEEE LU European Union customers Call your Customer Service representative for equipment pick up and recycling See www appliedbiosystems com for a list of customer service offices in the European Union Attune Acoustic Focusing Cytometer User Guide 151 Safety Labels on Instruments The following CAUTION WARNING and DANGER statements may be displayed on Applied Biosystems instruments in combination with the safety symbols described in the preceding section Hazard Symbol English Fran ais Z N CAUTION Hazardous chemicals Read the Safety Data Sheets SDSs before handling ATTENTION Produits chimiques dangereux Lire les fiches techniques de s ret de mat riels avant toute manipulation de produits CAUTION Hazardous waste Refer to SDS s and local regulations for handling and disposal ATTENTION D chets dangereux Lire les fiches techniques de s ret de mat riels et la r gulation locale associ es a la manipulation et l limination des d chets AN DANGER High voltage DANGER Haute tension WARNING To reduce the chance of electrical shock do not remove covers that require tool access No user serviceable pa
158. uidics bottles to prevent any bacterial contamination in the bottles Contamination symptoms include Bacteria growth in the bottle indicated by cloudy fluid globs or strings in the fluid or discoloration of the fluid A very high number of events that do not correspond to the sample i e sample dilution has no effect You can confirm by running Attune Performance Tracking Beads and seeing the high event rate over 1000 events sec compared to the expected 200 300 events sec To decontaminate the fluidics bottles sl 2 3 Disconnect all fluidics bottles from the instrument Discard all unused fluids Pour at least 20 mL deionized water in each bottle replace cap and invert or gently shake to coat all internal surfaces Discard deionized water Pour at least 20 mL 70 isopropanol in each bottle replace cap and invert or gently shake to coat all internal surfaces Discard isopropanol Invert each bottle and allow to air dry Place fresh Attune Focusing Fluid Attune Wash Solution and Attune Shutdown Fluid in the corresponding bottle Replace all fluidics bottles on the instrument Run Startup function ss Note All Attune fluids are packaged under sterile conditions If a 10X lt oe bh solution is used the quality of the deionized water used to dilute the 10X will affect the quality of the final working solution If contamination is observed in the 1X fluid discard fluid immediately and f
159. ular basis as listed below AN CAUTION BIOHAZARD All biological samples and materials that come into contact with them have the potential to transmit infectious diseases and are considered biohazardous Follow all applicable local state provincial and or national regulations Wear appropriate protective eyewear clothing and gloves Never pipette by mouth The table below lists the routine maintenance procedures that keep the Attune Acoustic Focusing Cytometer and all its peripheral systems in good working condition Visual inspection of sample injection Daily port Visual inspection of fluidics tanks and Daily connections Changing focusing fluid filter The frequency of maintenance depends on how often you run the cytometer Attune Acoustic Focusing Cytometer User Guide 45 Daily Maintenance Daily Shutdown Daily shutdown involves executing the Shutdown function This function ensures that all sample fluid and dyes have been removed from the fluidics lines and the two pumps have been decontaminated and filled with Attune shutdown solution to prevent the formation salt crystals The shutdown procedure takes approximately 30 minutes for the standard 30 rinse cycles but most of the steps are automated and under computer control At the end of the shutdown procedure the cytometer is automatically powered down Visual Inspection Visually inspect the sample injection port fluidics tanks and connections and the
160. um Bright Bead PMT Delta PMT Background Laser Actual MFI Voltage Voltage B Lin Reg Result Violet 6957 13 56 2825 75 mv NJA Criteria Met 64 Attune Acoustic Focusing Cytometer User Guide Levey Jennings Report Example The sample report below shows the Blue Violet configuration with the Delta PMT Voltage and Bright Bead HPCV values for FSC SSC BL1 BL2 BL3 VL1 VL2 and VL3 The report for the Blue Red configuration shows the Delta PMT and Bright Bead HPCV values for FSC SSC BL1 BL2 BL3 BL4 RL1 and RL2 Levey Jennings Report Instrument Name Your Instrument Name Operator Administrator Serial Number Your Instrument Serial Number Run Date 11 15 2010 3 41 PM Institution Your Institution Name Product Name 4 Peak Beads Software Version 1 2 0 Lot Number 756080 Expiration Date 2 11 2013 FSC Delta PMT Voltage FSC Bright Bead HPCV 100 gt 75 a o o 50 xa 2s S o 2 350 200 50 100 4 250 4 400 550 700 850 1000 Delta PMT mY Criteria Met Borderline Out Of Range No Data Page 1 of 4 BL1 Delta PMT Voltage a PMT mY e Delt BL2 Delta PMT Voltage Page 2 of 4 Attune Acoustic Focusing Cytometer User Guide 65 Levey Jennings BL3 Delta PMT Voltage BL3 Bright Bead HPCV Report Example continued gt U Q 2 g Delta PMT mY VL1 Delta PMT Voltage VL1 Br
161. une Acoustic Focusing Cytometer User Guide 75 then directed to the appropriate detector where it is translated into a voltage pulse proportional to the amount of fluorescence emitted All of the voltage pulses are recorded and can be presented graphically Multiple colors can be used on a flow cytometer and the number of colors that can be detected depends upon the number of detectors available in the cytometer The different colors are collected using select optical filters that direct the light to the right detector and capture the peak fluorescent signals Optical Filters Optical Filters separate the light scatter and fluorescence directed to detectors by wavelength which is measured in nanometers nm They selectively transmit light having a particular range of wavelengths while absorbing or reflecting the remainder photons reflected photons absorbed ec There are five types of optical filters used in flow cytometry e Bandpass filter BP e Longpass filter LP e Shortpass filter SP e Dichroic mirror DM e Neutral density filter ND 76 Attune Acoustic Focusing Cytometer User Guide Bandpass Filter Bandpass Filter BP is a device that passes wavelengths within a certain range and attenuates i e rejects wavelengths outside that range Combining an LP filter and an SP filter produces a bandpass BP filter These filters usually have lower transmittance values than SP and LP filters and block all waveleng
162. ution see page 11 for instructions on filling the fluid tanks Attune Acoustic Focusing Cytometer User Guide 13 Run Shutdown Function Perform System Flush Q IMPORTANT Powering the instrument on and off within 30 minutes can decrease the laser lifetime The Shutdown function powers off the laser and the instrument automatically If you interrupt the script you will need to force exit then restart the script and let it run to completion Log Out of the Attune Cytometric Software Click Shut Down The Shutdown prompt screen appears Select Standard for 10 wash cycles or Custom number of cycles to enter the desired number of wash cycles Click Next The software provides instructions to perform the shutdown operation The Attune Cytometric Software automatically performs the shutdown operation and the shutdown status window displays the shutdown function being executed At the end of the shutdown operation the Attune Cytometric Software automatically powers down the Attune Acoustic Focusing Cytometer Q IMPORTANT If you cancel the shutdown allow at least 10 minutes for the lasers to reach operating temperature before running any samples You will also need to re run Startup 1 IMPORTANT If you intend to leave the Attune Acoustic Focusing Cytometer in the shutdown state for longer than two weeks perform system flush and leave the instrument in deionized water to prevent salt crystals
163. v from the Beadlots file folder and return to step 5 ye Note The Beadlot file folder is located on C Programs Applied Biosystems Attune Performance Test Beadlots 06 Attune Acoustic Focusing Cytometer User Guide 7 Click Run Baseline Calculations to initiate the automated baseline calculations The Workspace displays the acquisition plots and events appear in the plots as the Attune Cytometric Software proceeds with baseline calculations Tors Attune Cytometric Software v2 1 Admin fo a e a ppl ied Collecting data for Analysis biosystems Stop 80 di Scatter Blue Laser Violet Laser FSC H SSC H BL1 H 10 10 j ae a 154 10 10 j 20 10 J 1 J z 31 0 a 43 213 2 1 ji E F 10 10 IE 8 A K an 10 J 4 10 ji 0 5 2 10 10 wa TTS 24 0 0 10 10 il my 0 1 Had a 0 10 o 10 10 00 05 10 15 20 00 05 10 15 20 10 10 10 10 4 r i 0 0 0 5 10 15 20 0 vu FSC H 1076 SSC H 106 BL1 H FSC 10 6 BL3 H 3 35 PM 5 17 2012 _ AT ma The progress bar provides an ongoing update during the run and the status display reports the continuing steps of the baseline calculation Note The baseline calculations take approximately 15 minutes to complete 8 At the end of the run the following message appears on the status displa
164. y DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations gt eS Except for IMPORTANT safety alerts each safety alert word in a Life Technologies document appears with an open triangle figure that contains a hazard symbol These hazard symbols are identical to the hazard icons that are affixed to Applied Biosystems instruments see Safety Symbols on page 151 Attune Acoustic Focusing Cytometer User Guide 149 Symbols on Instruments Electrical Symbols The following table describes the electrical symbols that may be displayed on on Instruments Applied Biosystems instruments Description Indicates the On position of the main power switch Indicates the Off position of the main power switch Indicates a standby switch by which the instrument is switched on to the Standby condition Hazardous voltage may be present if this switch is on standby Indicates the On Off position of a push push main power switch Indicates a terminal that may be connected to the signal ground reference of another instrument This is not a protected ground terminal Indicates a protective grounding terminal that must be connected to earth ground before any other electrical connections are made to the instrument Indicates a terminal that can receive or supply alternating current or voltage
165. y and the Baseline Calculations Report is displayed Attune Acoustic Focusing Cytometer User Guide 57 Baseline The Baseline Calculations Report screen summarizes the results and compares them with the target values It contains the cytometer and user information performance tracking beads lot information detector and laser settings specifications cytometer settings and results The sample report below shows the Blue Violet configuration with the parameters FSC SSC BL1 BL2 BL3 VL1 VL2 and VL3 The Blue Red configuration parameters are FSC SSC BL1 BL2 BL3 BL4 RL1 and RL2 Calculations Report n Attune Cytometric Software v2 1 Admin lle applied e biosystems Main Menu by Kafe technologies Current Baseline 5 17 2012 3 33 PM 756080 Baseline Report n Lot No 756080 H Page ija Q 125 Er Ej Find T Expiration Date 05 20 2021 Curren t Baseline _________________ Baseline 5 17 2012 3 33 PM 756080 Baseline Calculations Report Previous Baselines Baseline 12 14 2011 9 23 AM 756080 Instrumen t Name Operator Administrator Serial Number NA Run Date 5 17 2012 3 33 48PM Institution Product Name 4 Peak Beads Software Version 2 1 0 7869 Lot Number 756080 Laser Delay 435 Expiration Date 05 20 2021 Detector Settings Bright Bead BrightBead pmr Bight Quantum Background Laser Parameter Bead Efficiency Lin Reg Result Target MFI Actual MFI B

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