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BD FACSVantage SE Digital Option User's Guide

1. Sort by Parameter Sort by Formula Click OK 30 BD FACSVantage SE Digital Option User s Guide 13 Resize the statistics view to fill the remaining 1 3 of the worksheet Your worksheet should look similar to the example shown in Figure 2 1 Figure 2 1 Instrument QC worksheet 022306 488 nm SSC A 1 000 50 190 160 290 260 FITC A 1 000 60 190 160 290 260 60 100 150 200 022306 488 nm Count 10 250 50 100 150 200 250 FSC A x 1 000 FSC A x 1 000 a 022306 488 nm 022306 488 nm 100 150 200 250 FITC A amp 1 000 PA 022306 488 nm 2 022306 488 nm 15 15 Count 10 3 50 100 150 200 250 PE A x 1 000 TATI T ATT ATA 10 10 10 PerCP Cy5 5 A Experiment Name Feb 2006 Specimen Name 022306 Tube Name 488 nm Record Date OP GUID FSC A FSC A FITC A FITC A Population Events Parent Mean CY Mean cv HB all Events O HE WHE HH HHE ARE PE A PE A PerCP PerCP Mean ___CWY _ Mean cv HEE HARE GHAR HH Sheet1 from the Global Sheet menu The specified worksheet will appear when you move the current tube pointer to the 488 nm tube Chapter 2 Instrument Setup and Optimization 14 Select the 488 nm tube in the Browser in the Inspector choose Global 31 32 Optimizing Signals from the Primary Laser The following controls will be used to optimize signals from the primary la
2. NC V 27 Yield Mask Yield Mask target particle Figure 4 6 Target particle outside a Yield Mask of 8 trailing drop not sorted drop being interrogated leading drop not sorted Yield Mask Yield Mask target particle When the Yield Mask is set to zero only one drop the drop containing the target particle will be deflected when the mask is set to 32 two drops will always be deflected Yield Masks between 0 32 will sort either one or two drops When more than one drop is deflected in the same direction residual charge from the first drop will degrade the quality of the side stream Thus when four way sorting or sorting into small wells where precise deflection is required a Yield Mask of zero is recommended Chapter 4 Sorting 89 90 NOTICE Yield Masks cannot be used in conjunction with Phase Masks Thus when the Yield Mask is greater than zero the Phase Mask automatically reverts to zero Purity Mask The Purity Mask setting defines how close in 1 32 drop increments a contaminating drop can be located before ignoring the drop being interrogated For example when the Purity Mask is set to 16 the drop being interrogated will not be sorted if a non target particle falls within the first or last 8 32 of the leading or trailing drop In the following example a non target particle falls within the first 8 32 so the i
3. Breakoff values are used for the following Frequency adjusts the frequency of the drop drive from 1 0 102 0 KHz determines the number of drops formed per second Amplitude adjusts the amplitude or intensity of the drop drive from 0 0 80 0 V Phase adjusts the phase between drop generation and charging of the droplets from 0 360 degrees The selected value is sent to both the drop charging electrode and the drop strobe Drop delay sets the amount of time between when an event is measured and the breakoff point from 10 140 drops The drop delay value determines which drop will be deflected Drop sequence determines the sequence of test pulses Changes to the numerical field are not saved after restarting the application 78 BD FACSVantage SE Digital Option User s Guide Using the Streams Controls Stream charging and trajectory are adjusted using the controls in the Streams tab Enter a value to turn on the corresponding stream BreakOtt Far Left F5 a Yl 2nd Drop F9 il Left F6 40 Yl 3rd Drop F10 J 40 St Right F7 40 Y 4th Drop F11 s Ed Far Right F8 a YH e Far Left Left adjust the far left and left streams by changing the amount of charge applied to sorted droplets from 0 100 The far left stream is used for sorting onto a plate or slide and for four way sorting the left stream is used for two and four way sorting Right Far Right adjust the fa
4. 4 Optional Delete the APC Cy7 H histogram 5 Deselect the checkbox for the APC Cy7 H parameter 44 BD FACSVantage SE Digital Option User s Guide Recording and Analyzing Second Laser Results 1 Optional Adjust the voltages to display fluorescence signal between 100 000 and 150 000 Adjust the Sample Differential knob to decrease the event rate to approximately 200 events second Alt click the current tube pointer to record data for the 633 nm tube Alternatively click Record Data in the Acquisition Dashboard Copy the second laser results into your QC log Copy the fluorescence channel means into the QC log and print out the worksheet for your records Figure 2 8 Figure 2 8 Results for second laser optimization 041502 633 nm 041502 633 nm APC A 1 000 60 290 250 190 R lt pr a a A lt 50 100 150 200 250 50 100 150 200 250 FSC A 1 000 FSC A x 1 000 041502 633 nm 041502 633 nm APC CyT Count 180 290 390 4p0 sgo 690 790 Count 50 100 150 200 250 50 100 150 200 250 APC A 1 000 APC Cy7 A x 1 000 Experiment Name Feb 2006 Specimen Name 022306 Tube Name 633 nm Collection Date Feb 23 2006 10 19 21 AM APC A APC A APC Cy7 A APC Cy7 A Population Mean cv Mean cv BB I Events 133 509 15 6 134 462 16 5 Dd aPc 134 381 3 8 135 353 6 5 D APC Cy7 134 334 3 9 135 342 6 5 Chapter 2 Instrument Setup a
5. Fewer events than expected in gated population Events left out of gate When drawing a gate make sure events on the axis are included Plot zoomed Unzoom the plot or make the gate bigger Laser delay set incorrectly Adjust the laser delay settings See Optimizing Signals from the Second Laser Intercept on page 39 or Optimizing Signals from the Third Laser Intercept on page 46 Window extension set incorrectly Adjust the window extension Refer to the BD FACSDiva Software Reference Manual if needed Increasing threshold results in decreased area signal Window extension too small Slightly increase the window extension to maximize area signal A Increasing the window extension too much results in more electronic aborts or high CVs Area measurement off scale while the height measurement is on scale Area scaling too high Decrease area scaling to move the area measurement back on scale If necessary adjust area scaling to make the area measurement match the height measurement Chapter 7 Troubleshooting 149 Sorting Troubleshooting Observation Function keys not responding for sort setup values Possible Causes Sort Setup window inactive Recommended Solutions Select the window title bar to make it active and then press the required function key Defined population not listed in Add menu Population defined using snap to gate Redefine the po
6. 8 st Phase F3 0 Shi Drop Delay F4 10 00 2t Drop Sequence 10000200003000040000 standard pressure Streams Frequency F1 J 55 0 at Amplitude F2 o 8 at Phase F3 o o Shi Drop Delay F4 28 00 Hl Drop Sequence 10000200003000040000 high pressure t Test Sort for standard pressure 10 12 psi y x3 turn on Attenuation as well DropDrive TestSort Attenuation standard pressure only AA To prevent shock do not touch the nozzle when the drop drive is on or drops are being charged In digital mode the drop drive is on when the Drop Drive button shows a drop pattern in the Sort Setup window drops are being charged when the Test Sort button shows a drop pattern or when the Sort button has been clicked Chapter 4 Sorting 99 3 Adjust the frequency to obtain the optimal breakoff distance A When sorting with the 70 um nozzle tip at 10 12 psi the frequency should be in the range of 22 30 kHz For other nozzle sizes and sorting pressures see the following table Nozzle Size um Sheath Pressure psi Frequency kHz 50 11 15 32 34 70 10 12 22 30 100 8 9 15 20 When sorting with the 70 um nozzle tip at 35 psi the frequency should be in the range of 55 77 kHz For other sorting pressures see the following table Sheath Pressure psi Frequency kHz 50 60 65 99 30 50 55 77 20 30 40 55 Optimize these values for your sorting application In general a small
7. Click the tab to display Sheet1 When a tube is recorded the analysis is copied to the top most worksheet displayed After compensation calculation a compensation worksheet is usually the top most worksheet Switch to Sheet1 to copy the analysis to a blank worksheet To locate the worksheet click the left arrow control or press the arrow key on your keyboard until the worksheet is displayed Alternatively choose Worksheet gt New Worksheet to create a new blank worksheet BD FACSVantage SE Digital Option User s Guide 11 Click the Worksheets View button to return to the global worksheet view green tinted tabs Move the current tube pointer to the first unrecorded tube Install the corresponding tube on the cytometer and click Acquire Data View the data on the global worksheet make adjustments to gates as needed Adjustments will also apply to the next tube that is viewed on the global worksheet If you don t want to alter the global worksheet record the data and make adjustments on the tube specific worksheet Alt click the current tube pointer to record data Click Next Tube to move the current tube pointer to the next tube in the Browser Repeat steps 7 through 10 for the remaining tubes in the experiment To view data on a tube specific worksheet after recording double click the tube name in the Browser Chapter 3 Running Samples 71 72 BD FACSVantage SE Digital Option User s Guide 4 Sorting
8. You can program BD FACSDiva software to sort a specified number of particles from multiple populations into a variety of sorting devices including tubes plates and slides Specialized four way sorting hardware included with the option provides the ability to sort into four tubes simultaneously Up to four defined populations can be sorted into each tube allowing up to 16 populations to be sorted at one time Any subpopulation can be used for sorting including populations defined by quadrant gates interval gates or derived Boolean gates A single sort population can be defined by up to eight gates The following topics are covered in this chapter e Sorting Controls on page 74 e Conflict Resolution with BD FACSDiva Software on page 88 e General Sorting Overview on page 94 e Setting Up for Sorting Into Test Tubes on page 96 e Calculating the Drop Delay on page 104 e Sorting on page 107 e Setting Up for Sorting Into a Plate or Slide on page 109 73 Sorting Controls 74 The digital option provides four sets of sorting controls Controls on the BD FACSVantage SE control panel only shaded controls are active in digital mode Refer to the BD FACS Vantage SE User s Guide for a full description PEPE sa STREAM CONTROLS O O center stream control deflection plates on off VHOLD L drop strobe on off m D stream lamps on off VIEWING SORT CONTROLS MARK 30 O A A p
9. APC Cy7 A 1 000 APC Cy7 A 1 000 e Reset the window extension to the appropriate setting typically 2 A larger window extension allows more flexibility for capturing pulses Verifying Area Scaling for the Second Laser Because each laser has a different laser intercept height area scaling needs to be verified for each laser after its signal has been optimized Refer to the BD FACSDiva Software Reference Manual for more information about area scaling 1 In the Parameters tab select the height H checkbox for APC Cy7 2 Create a histogram for APC Cy7 H draw an interval marker around the fluorescent peak In the Laser tab of the Instrument window adjust area scaling for the second laser until the APC Cy7 A intensity is similar to the APC Cy7 H intensity Figure 2 7 on page 44 Chapter 2 Instrument Setup and Optimization 43 Figure 2 7 Second laser area scaling before left and after right adjustment Instrument xi be Toe Status Parameters Threshold Compensation Ratio Laser Status Parameters Threshold Compensation Ratio Laser Area Scaling 022306 633 nm_001 022306 633nm_001 APC Cy7 A Count 50 100 150 200 250 50 100 150 200 250 APC Cy7 A 1 000 APC Cy7 A 1 000 022306 633 nm_001 022306 633 nm_001 APC Cy7 H APC Cy7 H 50 100 150 200 250 50 100 150 200 250 APC Cy7 H 1 000 APC Cy7 H 1 000
10. CD19 APC A Mean Mean CD8 APC Cy A Mean Mean BB Lymphocytes H T cells LIT cytotoxic H T Helper E NK Cells H B Cells 8 588 15 796 13 369 6 636 15 410 861 155 18 023 27 423 19 393 42 17 262 17 885 12 471 67 90 15 465 13 902 Chapter 3 Running Samples 69 70 Saving the Analysis Now that the analysis strategy has been defined you can use it to analyze data for the remaining tubes in the experiment Global worksheets allow you to apply an analysis strategy to a series of data files either during recording or for a series of recorded tubes M Tip Use batch analysis to automatically process data files from a series of recorded tubes You can print worksheets or export statistics using options in the batch analysis dialog box When data is analyzed on a global worksheet the analysis will not be saved with each tube Do the following to automatically save a copy of the analysis with each tube as it is recorded 1 Choose Edit gt User Preferences and enable the preference to Save analysis after recording Optional To place each tube s analysis on a new worksheet also enable the Tube specific worksheet preference This preference automatically starts a new worksheet for each tube s analysis Refer to the BD FACSDiva Software Reference Manual for more information about user preferences Click the Worksheets View button to switch to the standard worksheet view grey tinted tabs
11. Calcium Flux 131 Setting Up the Experiment 1 Choose Instrument gt Instrument Configuration and verify the current configuration Make sure that the configuration lists the UV1 and UV2 parameters and that the lasers and channels correspond to the optical bench configuration M Tip To help keep track of UV parameters create a custom configuration with Violet in the UV1 and Blue in the UV2 parameter names Configuration D Current Configuration 6 color Configurations Parameters Configuration Modified Date Parameter Laser 211 6 06 6 01 00 AM FSC Detector FSC 7 color 2 16 06 6 01 00 AM SSC SSC 8 color 216 06 6 01 00 AM FITC FLA PE PerCP Cy5 5 uv uv2 FL2 FL3 F4 FL5 CSI 17 27 Gl d Gd T APC Add Set Duplicate Import Export OK LE A For accurate data results the instrument optics must match the current instrument configuration 2 Create a new experiment specimen and tube rename the experiment Calcium Flux M Tip To place the experiment inside an existing folder select the folder before creating the experiment 3 Rename the first tube Ca 1 This tube will be used to optimize signals from the UV 4 calcium Fux laser Your experiment should look similar to that shown in the figure at the right 132 BD FACSVantage SE Digital Option User s Guide 32 Instr Settings Global Worksheets Specimen _001 L
12. the BD logo BD Calibrite BD CellQuest BD FACS BD FACSDiva BD FACStation BD FACSVantage and BD TurboSort are trademarks of Becton Dickinson and Company FlowJo is a trademark of Tree Star Inc Microsoft and Windows are registered trademarks of Microsoft Corporation ModFit LT is a trademark of Verity Software House Inc All other company and product names might be trademarks of the respective companies with which they are associated This product is for Research Use Only Not for use in diagnostic or therapeutic procedures Patents PE and APC US 4 520 110 4 859 582 5 055 556 Europe 76 695 Canada 1 179 942 PerCP US 4 876 190 Cy5 5 and Cy7 US 5 268 486 5 486 616 5 569 587 5 569 766 and 5 627 027 PE Cy7 US 4 542 104 APC Cy7 US 5 714 386 FCC Information WARNING Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the user s authority to operate the equipment NOTE This equipment has been tested and found to comply with the limits for a Class A digital device pursuant to Part 15 of the FCC Rules These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instruction manual may cause harmful interference to radio communications Operation of this
13. value sets Choose a named setup from the drop down menu and then click OK PA Recall e Choose Instrument gt Sort Setup gt Delete to delete a predefined value set Choose a named setup from the drop down menu and then click OK 80 BD FACSVantage SE Digital Option User s Guide Sort Layout A Sort Layout is a floating window containing all sorting instructions and controls The Sort Layout designates which device will be used to collect sorted particles and which particles will be sorted into each sort location Up to four sort counters can be displayed in the window to give ongoing status during a sort Only one Sort Layout can be open at a time but you can create several layouts for a single tube as long as each Sort Layout has a different name Sort Layouts can also be added to global worksheets Sort Layouts are available for up to ten default collection devices additional custom devices can be defined See Creating a Custom Device on page 113 Figure 4 1 Default Sort Layout devices 2 Tube 4 Tube 6 Well Falcon 24 ell Costar 24 Well Falcon 48 Well Costar 60 Well Nunc 96 Well Standard Slide Frosted End Slide Standard Examples of Sort Layouts for different devices are shown in the following figures instructions for setting up a Sort Layout can be found in Setting Up the Experiment on page 108 Chapter 4 Sorting 81 Figure 4 2 Sort Layout for tubes top and for 48 well plate
14. 1 o F PerCP Cy5 5 1 FL3 Hoechst 33258 3 FL4 APC Cy7 2 FL6 APC 2 FL7 Add Set Duplicate Add FITC FL1 530 30 575 26 S Cc 712 12 610 SP S MQ PerCP Cy5 5 FL3 APC Cy7 FL6 42412 Hoechst 33258 FL4 L Appendix A Optical Configurations 155 APC FL7 O Q Seven Color Configuration Configuration D Current Configuration 6 color alternate Configurations Parameters Configuration Modified Date Parameter Laser Detector 6 color 413 06 9 44 00 AM IFSC 11 FSC ssc ld ssc 8 color 4413 06 9 44 00 AM FITC id FLA 6 color alternate 413 06 3 09 45 PM PE 11 FL2 PerCP Cy5 5 iu FL3 uvi l FL4 UVv2 8 FLS APC Cy7 2 FL6 APC 12 FL Add Set Duplicate FITC FL1 530 30 575 26 E PE FL2 War 560 SP 712 21 610 P MQ PerCP Cy5 5 FL3 395120 780 60 UV1 FL4 Ia D l Q APC Cy7 FL6 505 SP F AN T10 LP 485 22 S 660 20 UV2 FL5 ssc wi APC FL7 S 156 BD FACSVantage SE Digital Option User s Guide Eight Co
15. 127 Chapter 6 Calcium Flux 129 Intracellular Calcium Concentration Q Q eee e cec eee eens 130 Calcium Flux Optimization cece ee eens 131 Using the Time Parameter ococe coec e 131 Setting Up the Experiment occ ec cece eee ences 132 Optimizing the Calcium Sample QQ cece ee ee eee 135 Measuring Calcium Flux 0 0 ccc cece nen nes 137 Analyzing Data ste r A R A Le Raa AAS OG MAEN eS 139 Chapter 7 Troubleshooting 141 Electronics Troubleshooting 0 000s cece ecce cc 142 Acquisition Troubleshooting 0 cece cece eee een ences 144 Sorting Troubleshooting c eee cc 150 Contents vii Appendix A Optical Configurations 153 Six Color Contigutation dnanaris a Seve bx ieee ble dee eed Ne hoe odds 154 Alternate Six Color Configuration Five Colors DNA 155 Seven Color Configuration 0 000s cece eee tence een eens 156 Eight Color Configuration coco ceca 157 Alternate Eight Color Configuration eee eens 158 Configuration Worksheet 0 cece cece tent tenes 159 Index 161 viii BD FACSVantage SE Digital Option User s Guide About This Guide This guide describes how to operate the BD FACSVantage SE flow cytometer in digital mode using the digital option To familiarize yourself with BD FACSDiva software do the tutorials in Getting Started
16. 633 nm tube 40 BD FACSVantage SE Digital Option User s Guide Optimizing Signals from the Second Laser 1 Block the third beam intercept if present by closing the laser shutter Refer to the laser manufacturer s instructions Click to move the current tube pointer in front of the 633 nm tube and click Acquire Data Vv Tip If you do not see any signal increase the appropriate PMT voltage or change to Log If you still do not see any signal verify the Delay setting for the second laser step 6 on page 42 NOTICE During digital operation use the analog pulses displayed on the analog oscilloscope for troubleshooting purposes only Adjust the appropriate beam splitter s to direct the 633 nm laser signal to the appropriate PMT s Adjust the two rear rotators rear knobs to obtain the highest signal intensity and lowest CV on the fluorescence plots The knobs are located on the beam steering prism assembly of the laser being optimized If necessary adjust the two front translators front knobs to obtain the highest signal intensity and lowest CV Minor adjustment of the translators might be needed after performing laser alignment The translators do not need adjusting on a daily basis Chapter 2 Instrument Setup and Optimization 41 6 Optimize the Delay setting for the second laser Adjust the delay to synchronize laser signals in time The delay has been properly adjusted when the fluorescent signal intensity i
17. A vs PI W dot plot e PI A histogram 7 Create a statistics view and display the mean and CV for PI A and PI H e Right click the CEN tube and choose Create Statistics View e Click the Edit Statistics button in the Inspector On the Population tab deselect Events and Parent On the Statistics tab select the Mean and CV for PI A and PI H Figure 5 3 on page 120 e Set Decimal Places to 1 for the CVs Chapter 5 DNA Analysis 119 Figure 5 3 Setting up the statistics view Edit Statistics View Header Populations Al Median Geometri Meat Mi sD Mode FSC A E m m m H Ssc a m C C r r O C C P A E m m Vv wF m m m m PH D r d V F C C D C PW C C m O G m C C C Time m m O O C m C Decimal Places 0 o 2 7 o o o o 8 In the Acquisition Dashboard set the Events to Record to 10 000 evt and the Events to Display to 500 evt FB Acquisition Dashboard x Current Activity Active Tube ell Threshold Rate Stopping Gate Events Elapsed Time CEN 0 evtis 0 evt 00 00 07 Basic Controls gt Next Tube 5 Acquire Data Record Data tar Acquisition Setup a Storage Gate R an Everts Events To Record 10000 evt v Stopping Time sec 360 34 t Stopping Gate l AllEvents v Events To Display 500 evt lt M Tip Decreasing the number of displayed events will increase the data refresh rate Running CEN 1 Install the CE
18. Falcon 48 Well Costar Go to Home Set Home 60 Well Nunc Standard Slide Frosted End Slide Standard Test Sort button 4 Double click the Test Sort button to deposit a drop on the Home location 5 Carefully remove the collection device from the tray support and note where the drop was deposited 6 Wipe the collection device dry and place it back on the tray support 7 Adjust the Home location if necessary Click the appropriate arrow buttons to move the tray support as needed Large arrows move the tray by 5 steps small arrows move the tray by 1 step 8 Repeat steps 4 through 7 until the drop is centered appropriately 9 Click Set Home and then click Close 10 Proceed with Sorting on page 107 112 BD FACSVantage SE Digital Option User s Guide Creating a Custom Device You can program the robotic arm to sort into any grid configuration Create a custom device by entering the number of rows and columns and setting the Home and Farthest locations BD FACSDiva software calculates the increment between rows and columns to determine the sort locations 1 Choose Sort gt Custom Devices 2 Click Add in the Custom Devices dialog box A new device is added to the list of custom devices By default devices are named Custom Device_00x where x is the next consecutively numbered device Figure 4 20 Figure 4 20 Defining a custom device Custom Device_001 Name Custom Device_001 R
19. Mask of 16 trailing drop drop sorted leading drop T Phase Mask trailing drop drop not sorted leading drop L C Phase Mask Decreasing the Phase Mask to 8 allows more drops to be sorted However because the target particle is closer to the edge of the drop there is more variability in drop trajectory Figure 4 10 Figure 4 10 Sorted drop with Phase Mask of 8 trailing drop drop sorted leading drop il KI Mm M Phase Mask M Tip BD recommends using a Phase Mask of at least 8 when sorting single cells NOTICE Phase Masks cannot be used in conjunction with Yield Masks Thus when the Phase Mask is greater than zero the Yield Mask automatically reverts to zero Chapter 4 Sorting 91 Sort Precision Modes Mask values can be combined in many different ways By default five Sort Precision modes are already defined Purity 4 Way Purity Yield Single Cell Initial and Fine Tune Precision Mode Purity Yield Mask 32 Purity Mask 32 Phase Mask ro Single Cell j Add Close 4 Way Single Fine Precision Mode Purity Purity Yield Cell Initial Tune Yield Mask 32 0 32 0 32 0 Purity Mask 32 32 0 32 0 0 Phase Mask 0 0 0 16 0 0 Single Cell C C C O O e In Purity mode the Yield Mask
20. Up for Sorting Into a Plate or Slide on page 109 Setting Up for Sorting Follow the steps in this section to set up for sorting Each step is explained in more detail in the context of each sorting example 1 Install all sorting hardware including the appropriate nozzle tip for the size of the cells to be sorted For specific sorting hardware see Installing the Two Way Sorting Hardware on page 97 Installing the Four Way Tube Holder on page 97 or Installing the Sorting Hardware on page 110 AA Sorting hardware could be contaminated with biohazardous material Follow universal precautions when handling instrument hardware As a general guideline the nozzle tip should be six to ten times the particle diameter Perform daily instrument optimization and quality control each time the nozzle tip is changed 94 BD FACSVantage SE Digital Option User s Guide NOTICE Because the drop delay value cannot be lt 10 the digital option cannot sort with nozzle tips gt 100 um Use one of the following nozzle tips with the option Nozzle Tip Size um BD Catalog No 50 343592 60 343588 70 343593 80 343589 90 343591 1002 343594 a Not recommended for high pressure sorting gt 45 psi Start up the instrument and perform instrument optimization with appropriate sorting hardware installed See Instrument Optimization and Quality Control on page 26 Perform sample optimization for the sample to be sorted See Optimizing Setti
21. Vantage SE User s Guide Nozzle tip clogged or dirty Clean the nozzle tip as described in the BD FACS Vantage SE User s Guide Excessive amount of debris in plots Threshold channel too low Increase the threshold channel See Adjusting the Voltages and Threshold on page 59 Dead cells or debris in sample Examine the sample under a microscope Sample contaminated Re stain the sample making sure the tube is clean High CVs Instrument not aligned Verify the instrument alignment Event rate too high Decrease the event rate using the Sample Differential knob Poor sample preparation Repeat sample preparation Old or contaminated quality control QC particles Make new QC samples and perform the quality control procedure again Window extension too low Increase the window extension Cannot delete from Inspector Row not selected Select the row using the selection button 148 BD FACSVantage SE Digital Option User s Guide Acquisition Troubleshooting continued Observation High electronic abort rate gt 10 of system event rate Possible Causes Event rate too high Recommended Solutions Decrease the event rate Sample aggregated Filter the sample Sample too concentrated Dilute the sample Threshold channel too low Increase the threshold channel Window extension too high Decrease the window extension
22. be displayed only for two or four tube Sort Layouts Counters display the following information Sort Rate number of events second that met the sort criteria and were sorted Conflict Count number of events that met the sort criteria but were not sorted because of conflicts e Conflict Rate number of conflicts second Efficiency number of sorted events sort conflicts sorted events x 100 86 BD FACSVantage SE Digital Option User s Guide Monitoring a Sort During sorting each sort location field displays the number of actual sorted events When a target number is specified the field displays the actual number of events along with the number of target events A progress bar appears behind the Sort Rate counter field showing the progress of the sort zi TBNK_001 Sort Layout_001 x Device Precision Target Events Save Conflicts Pome Meta socor T Left Right P2 580 5000 OF3 290 5000 Sort Rate 80 evtis 40 evtis Confl Crit 4710 evt 4420 evt 3 80 evtis 40 evtis 100 100 H Pause View Counters Sort Report Choose Sort gt Sort Report to view a report of the current Sort Layout This menu item is enabled only if a Sort Layout is open and the instrument is not sorting The Sort Report can be printed or exported NOTICE After closing a Sort Layout all counter information is lost Thus you should print a Sort Report immediately aft
23. bottom Device Precision Target Events Save Conflicts collection _ s Tune pty device sort location field for far right tube sort counters Efficiency NA NA sot Brace sorting controls za Sort cila Sort Layout Precision Target Events Save Conflicts 48 Well Costar v singte Cell aas v pz sort location field for single well View Counters Figure 4 3 Sort Layout for frosted slide Sort Sample Sort Layout x Device Precision Target Events Save Conflicts slide Frosted End w Single Cell X jx sort location field for spot on slide View Counters 82 BD FACSVantage SE Digital Option User s Guide Setting Up a Sort Layout NOTICE If a global worksheet is displayed in the Worksheet window when you create a new Sort Layout the Sort Layout will be added to the global worksheet rather than the tube To ensure the Sort Layout applies only to the current tube switch to the Worksheet view before you create the Sort Layout 1 Right click on a tube in an open experiment and choose New Sort Layout Alternatively select a tube in the Browser and click the Sort Layout button in the Workspace toolbar E In the Sort Layout window choose the type of device from the Device menu Default sorting devices are listed along with any
24. defined custom devices The Sort Layout window changes depending on the selected device the number of rows and columns in the window matches the number of tubes wells or spots in the collection device Choose the Sort Precision mode from the Precision menu For more information see Sort Precision Modes on page 92 Enter the number of events to be sorted in the Target Events field Once defined the number of events can be reused by choosing from the drop down menu For continuous sorting choose Continuous from the Target Events menu Select the field s corresponding to the tube s well s or spot s where the population will be sorted and choose a defined population from the Add menu NOTICE Populations defined by Snap To gates and those derived from them cannot be sorted After you click in a sort location field a menu appears where you can choose to add delete or clear all populations in the field Figure 4 4 on page 84 Chapter 4 Sorting 83 Figure 4 4 Adding populations to be sorted Sort Sample Sort Layout xj TE T Target Events Save Conflicts brws x Tube v Puty Continuous Y 5 P1 Continuous oO P2 Ea Rate Delete gt Wr Confl Cnt Clear All ies Confl Rate gt MET Efficiency NA NA H Sort H reuse View Counters After you add a population the population and the number of target events are written to the corresponding sort location field M Tip Se
25. each tab make adjustments using the software controls or your keyboard To adjust a setting click in the field containing the value you want to change or press the indicated function key eg F2 for Amplitude NOTICE To use the function keys the Sorting window must be active highlighted Do one of the following to change any value e Select the value in the field and enter a new value e Click the slider button next to the arrow keys to access a slider control Click the pointer in the slider bar and drag it to a new value FA Medium x BreakOtt streams 360 Frequency F1 27 0 JH 288 Amplitude F2 20 0 H me Phase F3 20 l Drop Delay F4 45 00 2il ay Drop Sequence 10000200003000040000 72 slider control X i Af Drop Drive Test Sort Attenuation 76 BD FACSVantage SE Digital Option User s Guide Use the mouse to click the up and down arrows or press the arrow keys on your keyboard to increase or decrease the values in small increments e Hold down the Control key while clicking the arrows or pressing the keys to increase the increments of the values Using the Sort Setup Buttons The sort setup buttons control instrument functions for setting the breakoff point and setting up the streams thus the buttons are accessible from both tabs Click the button to turn on or off the respective instrument function Drop Drive turns the drop drive on or off When the drop
26. from the first laser beam This section describes how to optimize signals for a standard laser configuration primary intercept 488 nm secondary 633 nm third UV Refer to your BD FACS Vantage SE User s Guide for more information about how the optical bench is configured System configuration can vary greatly If your system has a half mirror in the OBS2 position Figure 2 5 use this procedure to optimize the signal detected by detector option 1 DO1 or detector option 2 DO2 located to the left of the half mirror If your system has a triple laser beam splitter in the OBS2 position use this procedure to optimize the signal detected by DO1 or DO2 located to the right of the triple laser beam splitter Figure 2 5 Detection of signals from the second laser intercept FL1 O FLI SG Ob OBS2 triple laser beam splitter S DO5 OBS3 IOI pe DO D TH 0882 hatt mirror o3 Q J DO 0021 OBS4 S OBS3 ssc QO ssc Q D04 S DO2 one additional laser two additional lasers FL3 ii ol Q NOTICE Your
27. is free of contaminating particles The digital option accurately measures particle position to within 1 32 of a drop Mask settings determine how drops are deflected when sorting conflicts occur There are three mask settings Precision Mode purty each of which addresses a different type of conflict eld Mask 2 Purit k 32 These settings are combined to define Sort Precision eL Phase Mask 0 modes each mode is made up of a set of masks 7 7 Precision modes are defined in the Sort Precision dialog Add Close box accessed from the Sort menu BD FACSVantage SE Digital Option User s Guide Yield Mask The Yield Mask setting defines how close to the edge of the drop in 1 32 drop increments a particle of interest can be located before sorting an additional drop Half of each Yield Mask setting defines an equal area at each end of the drop For example when the Yield Mask is set to 16 and an event is within 8 32 from the beginning of a drop the previous leading drop will be sorted If an event is within 8 32 from the end of a drop the following trailing drop will be sorted See Figure 4 5 Figure 4 5 Target particle within a Yield Mask of 16 trailing drop sorted drop being interrogated leading drop not sorted MA Mi If the Yield Mask were set to 8 for the same target particle the target particle would fall outside of the Yield Mask thus no additional drops would be sorted See Figure 4 6
28. log QC data should be analyzed for trends over the past 30 60 runs NOTICE QC results are affected by laser and fluidics performance BD Biosciences strongly recommends following the laser and fluidics maintenance procedures in the BD FACS Vantage SE User s Guide A Do not place heavy objects or lean on the instrument console while performing this procedure Unnecessary pressure on the instrument during or after instrument optimization could disrupt alignment and it would have to be performed again Preparing the Alignment Sample Choose an alignment sample that gives a consistent signal and is readily available such as chicken red blood cells CRBCs or alignment beads Make sure the alignment sample can be excited by your system s lasers and that the appropriate filters are installed to detect the alignment signal s Prepare the alignment sample according to the manufacturer s instructions BD FACSVantage SE Digital Option User s Guide Setting Up the Experiment The steps in this section show you how to set up an experiment for instrument optimization If you have already created a similar experiment you can reuse it by saving it as an experiment template Refer to the BD FACSDiva Software Reference Manual for information 1 A Choose Instrument gt Instrument Configuration and verify the current configuration Make sure the configuration lists the parameters to be measured and that the channels correspond to the opt
29. onto the cytometer 2 In the Acquisition Dashboard click Next Tube and then Acquire Data Alternatively move the current tube pointer to the next tube and click the pointer to start acquisition 3 Click Record Data or Alt click the current tube pointer to record data 4 When recording is finished install the next stained control tube onto the cytometer 5 Repeat steps 2 through 4 until data for all stained control tubes has been recorded 6 Double click the first stained control tube FITC stained control to display the corresponding worksheet 7 Verify the snap to interval gate surrounds the fluorescence positive peak on the histogram Figure 3 6 Adjust the gate if needed Figure 3 6 Gating the positive population FITC Stained Control FITC Stained Control SSC A 1 000 50 100 150 200 260 50 100180200280 FSC A x 1 000 8 Repeat steps 6 and 7 for the remaining compensation tubes 62 BD FACSVantage SE Digital Option User s Guide 9 Choose Instrument gt Instrument Setup gt Calculate Compensation If the calculation is successful a dialog box appears where you can enter a name for the compensation setup 10 Enter the name of your experiment as the setup name and click OK Compensation calculation has completed successfully Name 5 Color Expt 042804 M Tip To keep track of compensation setups include the experiment name date or both in the setup name NOTICE BD Biosciences
30. population 7 Copy the results into the QC log and print the worksheet for your records Keep a record of the primary laser results for future reference See the following figure for an example Figure 2 4 Primary laser optimization using CRBCs 022306 488 nm 022306 488 nm SSC A 1 000 PE A 1 000 50 100 150 200 250 50 100 150 200 250 FSC A 1 000 FSC A 1 000 022306 488 nm 022306 488 nm SC p1 Count 150 200 250 100 150 200 250 FSC A x 1 000 FITC A x 1 000 022306 488 nm pe 022306 488 nm PerCP Cy55 E 3 o 50 100 150 200 250 102 10 10 10 PE A c 1 000 PerCP Cy55 A Experiment Name Feb 2006 Specimen Name 022306 Tube Name 488 nm Collection Date Feb23 2006 10 52 52 AM FSC A FSC A FITC A FITC A PE A PE A PerCP C PerCP C Population Mean Cv Mean Cv Mean Cv Mean Cv EX FSc p1 453143 175 61 8219 944 503166 945 704 9 90 4 EX FSC p2 157 179 68 129 1327581 17 4 127 8293 17 4 1 788 2 20 8 KI FITC 151 515 8 23 2 135 154 1 13 1 130 175 5 13 2 1 812 3 18 0 M PE 150 960 8 23 5 134 032 9 14 4 129 111 8 14 4 1 798 3 19 0 Dd PerCP Cy55 150 292 0 23 9 1325215 17 3 127 7057 17 2 1 787 1 19 7 38 BD FACSVantage SE Digital Option User s Guide Optimizing Signals from the Second Laser Intercept If you are using more than one laser you will need to optimize signals from the second laser third laser or both lasers after optimizing signals
31. recommends that you always visually and statistically inspect automatically calculated overlap values The means of the positive controls should be aligned with the means of the negative Recording and Analyzing Data Once you have optimized the instrument electronics for your sample type you are ready to record and analyze data During analysis recorded data is displayed in plots and gates are used to define populations of interest BD FACSDiva software analyzes the gated data and calculates statistics that you can print or export With global worksheets data can be shown for a series of tubes on the same worksheet manually or in an automated batch analysis The following sections describe how to use BD FACSDiva software features to record and analyze sample data As an example data will be recorded and analyzed for two samples stained with the following reagents CD4 FITC CD16 CD56 PE CD3 PerCP Cy5 5 CD19 APC CD8 APC Cy7 Chapter 3 Running Samples 63 Setting Up the Global Worksheet This section shows you how to use a global worksheet to preview and record data for multiple samples 1 Create a new specimen rename the specimen LWB 2 Create two tubes under the LWB specimen rename the tubes appropriately For example T B NK_001 and T B NK_002 3 Create a global worksheet rename the worksheet Record Data To switch to the global worksheet view click the Worksheets View button in the Worksheet toolbar e If Defa
32. tube pointer next to the unstained control tube and click Acquire Data Adjust the FSC and SSC voltages to appropriately display the scatter properties of the LWB sample Figure 3 3 Figure 3 3 Voltages adjusted Unstained Control 100 150 200 250 FSC A x 1 000 Click the Threshold tab and adjust the FSC threshold if needed Set the threshold to remove most of the debris without cutting off the lymphocyte population Figure 3 3 Adjust the P1 gate on the Unstained Control worksheet to surround only the lymphocyte population Figure 3 3 Select the gate by clicking on its boundary Once selected you can drag the gate to move it or drag any of the selection handles to change the size and shape of the gate Chapter 3 Running Samples 59 7 Right click the P1 gate and choose Apply to All Compensation Controls The P1 gate on each Stained Control worksheet is updated with your changes 8 Select all fluorescence histograms on the Unstained Control worksheet 9 Inthe Plot Inspector select the Grid checkbox Figure 3 4 Figure 3 4 Plot Inspector for fluorescent plots Inspector x Pict Title Labels Tube n Controls Unstained Control X Parameter FITC A Y Parameter Esc a Biexponential Display l X Axis Plot Elements V Plot Outline V Tick Labels Background Color V Grid Outline V Tick Marks H C Full Half Population Draw BB l Events V es 1 In a four log display val
33. 3 through 9 138 BD FACSVantage SE Digital Option User s Guide Analyzing Data M Tip Data can also be exported for analysis in a third party application such as FlowJoTM 1 To better visualize the cellular response draw a series of interval gates on the time vs ratio dot plot Figure 6 5 Figure 6 5 Calcium flux data points over time Response E Resolution 70 Ratio UV1 A UY2 441 000 25 x 1 000 Specimen Name Specimen 1 Tube Name Ca CD3 stimulus 1 1 1 1 Collection Date Feb23 2006 7 03 56 PM UV1 A UV2 A Ratio UV1 A UV2 Population Mean Mean Mean All Events 50 827 80 018 Lymphs 41 060 59 942 Background 45 819 91 058 Response 50 014 78 407 Resolution 56 295 81 385 Tube Ca CD3 stimulus 1 1 1 1 Population Events Parent Total all Events 324 528 100 0 E Lymphs 229 779 70 8 708 Dd Background 15 826 4 9 4 9 Dd Response 72 552 22 4 224 D Resolution 40 581 12 5 12 5 Chapter 6 Calcium Flux 139 140 BD FACSVantage SE Digital Option User s Guide 7 Troubleshooting The tips in this section are provided to help you troubleshoot issues that might arise when using the digital option For instrument specific troubleshooting refer to the BD FACSVantage SE User s Guide for software specific troubleshooting refer to the BD FACSDiva Software Reference Manual or the software online help If additional assistance is required contact your local BD Biosciences technical support represent
34. 5 sorting 85 107 stopping sorting 85 107 streams adjusting 33 74 79 Streams tab 79 troubleshooting 151 turning on 77 T Target Events 83 technical assistance xi Test Sort 77 test tubes sorting into 96 Theta control 32 Index 167 third laser adjusting delay 48 optimizing signals 46 47 results 51 verifying area scaling 49 threshold adjusting 59 troubleshooting 149 Time parameter 131 troubleshooting acquisition 144 CVs 148 electronic aborts 149 electronics 142 event rate 146 147 Inspector 148 keyboard keys 76 150 low area signal 145 oscilloscope 150 plots 144 146 148 populations 149 150 results 151 scatter parameters 148 signals 145 149 sort conflict rate 150 sort counters 150 sort rate 150 sorting 150 streams 151 tubes 151 window extension 149 tubes compensation 57 four way sorting 97 98 label specific 57 sorting into 96 troubleshooting 151 two way sorting 97 two way sorting hardware 97 typographical conventions x U upgrading firmware error 142 user preferences 70 V viewing CellQuest Pro 20 global worksheets 70 voltages adjusting 59 W waste emptying 24 window extension adjusting 42 48 troubleshooting 149 worksheet configuration 159 worksheets viewing 70 workspace view options 20 workstation about 20 xX X control 32 Y Y control 32 Yield Masks about 89 Phase Masks using with 90 91 Yield mode 93 Z Z control 32 Z distance 33 168 BD FACSVant
35. 50 200 250 PIA 1 000 CEN Feb 23 2006 CV lt 3 9 Verify that the FL1 FL2 iris is open and check the CV of the Singlet population e If the CV is lt 3 continue with step 10 e If the CV is gt 3 skip to the following section then return to step 10 to finish this section 10 Click Record Data to save the data 11 Check the linearity and print the worksheet Note the means of the Singlet and Doublet populations Divide the mean of the Doublets by the mean of the Singlets The Doublet Singlet ratio should be 2 00 0 05 If you cannot achieve a ratio from 1 95 to 2 05 contact BD Customer Support Copy the means CVs and the calculated linearity result into the QC log 12 Remove the CEN sample from the cytometer 122 BD FACSVantage SE Digital Option User s Guide Optimizing the CV of the Singlet Population Perform the steps in this section only if the CV of the Singlet population is gt 3 After optimizing the CV return to step 10 in the previous section and finish the remaining steps in that section 1 Adjust the event rate to approximately 1 000 events second 2 Adjust the instrument optimization controls to obtain the lowest possible CV for the Singlet population e Ensure that the Display is set to 100 500 events e Close the FL1 FL2 iris e While viewing the PI A vs PI H plot adjust the Y control Excitation Beam Focus wheel X control Fluorescence Focus control
36. CSDiVa 3 Dit 4 gt ption L digital control switch Digital Oscilloscope The digital oscilloscope displays the digital drop drive waveform and the digital drop charges Figure 1 3 on page 17 Use the digital oscilloscope to verify charging of the side streams and the amplitude level Refer to the documentation provided with the oscilloscope for oscilloscope adjustments 16 BD FACSVantage SE Digital Option User s Guide Figure 1 3 Pulses displayed on the digital oscilloscope MEASURE digital amplitude AANA Wl i ii drop charge J p g vi ca Freq WEN Period voLTSIDIV VOLTSIDIV SECIDIV ake iby e cH 4 Nore Mg a Accudrop Monitor The Accudrop monitor shows an image of the streams illuminated by the Accudrop excitation source a diode laser Use the monitor to accurately set the drop delay and set up the streams for sorting as described in Calculating the Drop Delay on page 104 Refer to the BD FACS Accudrop User s Guide for specific information about the Accudrop option Digital Control Switch The digital control switch Figure 1 2 on page 16 determines how the instrument electronics are controlled When switched to On the instrument is in digital mode and the electronics ar
37. Click Sort in the Sort Layout window 6 Optimize the drop delay In the Breakoff tab in the Sort Setup window adjust the drop delay setting until most of the beads are in the left stream and the least are in the center stream Figure 4 17 using a 1 drop increment Before you adjust the drop delay the beads will appear as a bright spot on the center stream and a faint spot on the left stream Adjust the drop delay until the spot on the left stream is as bright as possible This will yield the most accurate drop delay Figure 4 17 Viewing beads on Accudrop monitor before left and after right adjustment left stream left stream center stream center stream 106 BD FACSVantage SE Digital Option User s Guide 10 11 Click Sort to stop sorting In the Sort Layout change the Precision mode to Fine Tune ma AccuDrop Beads Sort Layout_001 x Device Precision Target Events Save Conflicts 2 Tube wv Fine Tune ne bf a Left Right E AccuDrop B Continuous Repeat steps 5 through 7 adjusting the drop delay in a 0 1 drop increment The final drop delay setting is the optimal setting for the sort Move the emission filter away from the camera remove the tube from the cytometer Proceed with sorting See the following section Sorting Before beginning the sort do the following Perform sample optimization with the drop drive on and the frequency at an appropriate level Use ga
38. Create Compensation Controls Fluorophore Label FITC Generic PE Generic PerCP Cy5 5 Generic APC Generic APC Cy Generic Add OK Cancel Add label specific controls when your experiment contains samples stained with the same fluorophore conjugated to different antibodies labels that require different compensation values This is especially noticeable in tandem conjugates due to lot to lot variation Refer to the BD FACSDiva Software Reference Manual for more information A Compensation Controls specimen is added to the experiment along with a stained control tube for each compensation control Expand the specimen to view all tubes Worksheets containing appropriate plots are added for each compensation tube Compensation Controls 33 Instr Settings J Unstained Control FITC Stained Control PE Stained Control PerCP Cy5 5 Stained Control APC Stained Control apc cy7 Stained Control eee H 58 BD FACSVantage SE Digital Option User s Guide Adjusting the Voltages and Threshold The unstained control will be used to check for nonspecific antibody binding to adjust forward scatter side scatter and FSC threshold to gate the population of interest lymphocytes in this case and to adjust fluorescence settings 1 2 Install the unstained control tube on the cytometer Expand the Compensation Controls specimen in the Browser Click to set the current
39. D8 APC Cy7 plot and name the population T Cytotoxic Because the T cell population is selected first the T cytotoxic cells become a subset of all T cells Select the T cell population in the population hierarchy view draw a region around the double positive population on the CD3 PerCP Cy5 5 vs CD4 FITC plot and name the population T Helper Print the analysis See Figure 3 8 on page 69 for an example BD FACSVantage SE Digital Option User s Guide Figure 3 8 Lymphocyte analysis 3 16 56 45 4 1 9 8 All Events 100 150 200 280 FSC H x 1 000 3 16 56 45 4 1 9 8 Lymphocytes NK Cell 194 g 10 wW a w wo a 3 w 2 a 3 lt 2 a a s 370 10 10 10 10 CD3 PerCP Cy5 5 A 3 16 56 45 4 1 9 8 Lymphocytes 3 16 56 45 4 1 9 8 Lymphocytes CD19 APC A 102 109 10t CD3 PerCP Cy5 5 A 370 19 T Cytotoxic k T Helper 194 CD8 APC Cy7 A 193 CD4 FITC A 370 10 10 10 108 CD3 PerCP Cy5 5 A 370 102 102 10 108 CD3 PerCP Cy5 5 A Tube 3 16 56 45 4 1 9 8 Population BB All Events H Lymphocytes T Cells HEI T Cytotoxic fl T Helper NK Cells B Cells Experiment Name 6 color gating_001 Specimen Name Tube Name Lymphocytes 311 6 56 45 4 1 9 8 CD3 FITC A Population Mean CD16 CD56 PE A CD45 PerCP Cy 10 000 3 373 1 816 402 1 289 1 222 199 Record Date SOP Feb 23 2006 8 23 13 AM Administrator
40. Digital data is processed by BD FACSDiva software on a Microsoft Windows workstation analog data is processed by BD CellQuest Pro software on a BD FACStation Data Management system The computers are networked via an ethernet hub to share a printer Refer to the BD FACSDiva Software Reference Manual for information on how digital signals are measured BD FACSVantage SE Digital Option User s Guide Figure 1 1 BD FACSVantage SE flow cytometer with digital option installed Components For a description of digital option components see the following e Digital Electronics Module on page 16 e Four Way Sorting Tube Holder on page 19 e BD FACSDiva Workstation on page 20 Chapter 1 Features of the Digital Option 15 Digital Electronics Module The digital electronics oscilloscope BD FACS Accudrop monitor and digital control switch are housed within the digital electronics module Figure 1 2 The electronics are adjusted by your field service engineer during installation and do not require user maintenance Figure 1 2 Digital electronics module OOOO digital oscilloscope Accudrop monitor Se ee ed i FA
41. Drop Drive amplitude 146 BD FACSVantage SE Digital Option User s Guide Acquisition Troubleshooting continued Observation Unexpectedly low event rate Possible Causes Memory full Recommended Solutions Compare the processed event rate in BD FACSDiva software with the threshold counter on the instrument If the BD FACSDiva event rate is much lower quit and then restart the application Threshold channel too high Adjust the threshold channel See Adjusting the Voltages and Threshold on page 59 Sample not adequately mixed Mix the sample to suspend cells Sample too dilute Concentrate the sample Erratic event rate Sample aggregates Filter the sample Sample tube cracked Replace the sample tube Sample tube O ring worn Replace the O ring Refer to the BD FACSVantage SE User s Guide Nozzle tip clogged Clear the nozzle tip as described in the BD FACS Vantage SE User s Guide Sample contaminated Re stain the sample making sure the tube is clean Sheath tank low Fill the sheath tank Chapter 7 Troubleshooting 147 Acquisition Troubleshooting continued Observation Distorted scatter parameters Possible Causes Instrument settings adjusted incorrectly Recommended Solutions Optimize the scatter parameters See Adjusting the Voltages and Threshold on page 59 Air bubble Remove the air bubble Refer to the BD FACS
42. J ca1 Click the Parameters tab in the Inspector and make the following changes e Delete all parameters except FSC SSC UV1 and UV2 e Verify that the Log checkbox is deselected for all parameters Instrument xj Status Parameters Threshold Compensation Ratio Laser Parameter Voltage Log H Ww FSC 250 riel reici SSC 300 C IRFI C RC uv 500 O 4 UVv2 500 O BM O Click the Ratio tab and click the Add button choose UV1 A for the numerator and UV2 A for the denominator Instrument x Status Parameters Threshold Compensation Ratio Laser Numerator Denominator Scaling UY1 A uv2 a 25 00 Create the following dot plots for the Ca 1 tube e FSC A vs SSC A e UV1 A vs UV2 A e Time vs Ratio UV1 A UV2 A Show the tube and population names in the plot titles Select all plots on the worksheet click the Title tab in the Inspector and select the appropriate checkboxes Figure 6 2 on page 134 Chapter 6 Calcium Flux 133 Figure 6 2 Showing tube and population names in plot titles Inspector x Plot abels Dot Plot r Title Content I Specimen IV Tube IV Populations Custom Title r Title Font Face SansSerif v Size 12 Color H C taic Bold 8 Create a statistics view and display the mean for the UV parameters and the ratio e Right click the Ca 1 tube and choose Create Statistics View e Select the s
43. N sample tube on the cytometer turn the Fluidics Control knob to Run 2 Verify that the current tube pointer is next to the CEN tube in the Browser click the pointer to start acquisition Events appear in the plots 120 BD FACSVantage SE Digital Option User s Guide 3 Adjust the Sample Differential knob to obtain an event rate of approximately 1 000 events second The event rate is displayed in the Acquisition Dashboard 4 Adjust the FSC and SSC voltages to place the CEN on scale in the FSC vs SSC dot plot Figure 5 4 FSC and SSC voltages adjusted DNA QC Kit CEN 150 200 250 FSC A x 1 000 5 Adjust the PI voltage to place the singlet nuclei at approximately channel 50 x 103 on the PI A axis in the PI A histogram plot 6 While viewing the PI A vs PI H plot adjust the Y control and the Excitation Beam Focus wheel to maximize the signals Figure 5 5 Optimized PI A and PI H signals DNA QC Kit CEN 150 200 250 PLA x 1 000 7 Decrease the event rate to approximately 200 events second Chapter 5 DNA Analysis 121 8 Draw an interval gate around the first two peaks on the PI A histogram name the populations Singlets and Doublets Figure 5 6 Figure 5 6 Defining Singlet and Doublet populations DNA QC Kit CEN iz l Doublets Experiment Name DNA Tube Name Specimen Name DNA QC Kit Collection Date PI A PI A Population Mean H All Events 123 858 DJ singlets 48 134 EX Doublets 96 722 1
44. Red 3 FLS APC Cy7 2 FLG APC 2 FLT PE Tx Red 1 FL8 Add Set _Dupicate Add FITC FL1 530 30 575 26 E PE FL2 560 SP PE Tx Red FL8 610 20 S 640 LP 740 LP or 780 60 610 P I M PE Cy7 FL3 450 20 780 60 or 740 LP Hoechst Blue FL4 D Q APC Cy7 FL6 CEA 610 SP 710 LP 675 LP S 660 20 Hoechst APC FL7 SSC Red FL5 C For more information see Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo Goodell MA Brose K Paradis G et al J Exp Med 1996 18331797 1806 158 BD FACSVantage SE Digital Option User s Guide Configuration Worksheet Configuration Name 7 r AT S A ais JO es 45 Appendix A Optical Configurations 159 160 BD FACSVantage SE Digital Option User s Guide Index Numerics 4 Way Purity mode 92 A aborts electronic 149 See also conflicts sort AccuDrop determining dro
45. Sorting Into a Plate or Slide This section describes how to set up for sorting into a plate or slide Use this procedure as a guide to set up similar sorting experiments For general guidelines see General Sorting Overview on page 94 Chapter 4 Sorting 109 Installing the Sorting Hardware NOTICE For more details on hardware installation refer to the BD CloneCyt Plus User s Guide 1 Turn off the deflection plates A To prevent shock do not touch the deflection plates when the red warning light appears on the control panel 2 Remove the center stream aspirator and replace it with the right and center stream aspirator supplied with the BD CloneCyt Plus option 3 Install the metal ground shield and the plastic tray shield 4 Install the tray support on the support arm using the metal thumbscrew NOTICE Install only the shorter tray support provided with the digital option Previous versions of the tray support do not allow sufficient clearance between the plate and the sort chamber 5 Place a 96 well plate on the tray support Figure 4 18 Figure 4 18 Sorting hardware for a 96 well plate right and center stream aspirator EXCITATION BEAM FOCUS 110 BD FACSVantage SE Digital Option User s Guide Adjusting the Home Location When sorting into a 96 well plate or onto a slide the robotic arm holding the tray support is pre programmed to move a set interval betwe
46. age SE Digital Option User s Guide
47. ate plots for previewing the data For example create FSC vs SSC FITC vs PE PerCP Cy5 5 vs PE and APC vs APC Cy7 dot plots M Tip Double click the Dot Plot button to keep the button selected until you create all plots Chapter 3 Running Samples 65 Recording Data 1 2 w oO UU A Install the first sample tube onto the cytometer Move the current tube pointer to the first tube and click Acquire Data While data is being acquired draw a gate around the lymphocytes set the other plots to show data from the Lymphocyte population Alt click the current tube pointer to record data When all events have been recorded remove the tube from the cytometer Install the next sample tube onto the cytometer Move the pointer to the corresponding tube in the Browser and click the pointer to start acquisition Preview the data in the global worksheet Alt click the pointer to record data Repeat steps 5 through 8 until data has been recorded for all tubes Optional Print the experiment level instrument settings Right click the instrument settings icon and choose Print 66 BD FACSVantage SE Digital Option User s Guide Analyzing Data This section describes how to set up plots gates and a statistics view to analyze the recorded data By the end of this section your analysis should look similar to that shown in Figure 3 8 on page 69 1 Create a new global worksheet rename the worksheet T B NK Analysis 2 Create the fo
48. ative See Technical Assistance on page xi Troubleshooting suggestions in this chapter are grouped under the following headings e Electronics Troubleshooting on page 142 e Acquisition Troubleshooting on page 144 e Sorting Troubleshooting on page 150 141 Electronics Troubleshooting Observation Possible Causes Instrument Disconnected Power switched off on digital in Instrument window electronics module Recommended Solutions Check the digital oscilloscope If the screen is blank switch on the Digital control switch Communication failure between workstation and instrument e Quit the software and then restart it e If restarting does not work reset the digital electronics by switching off the power switch on the back of the digital electronics module and then switching the power back on Restart the computer Ethernet cable disconnected between workstation and instrument Unplug and then plug in the cable connectors and make sure they are secure IP address changed Enter the correct IP address Call BD Biosciences for assistance Upgrading firmware Firmware loading incomplete in Instrument window Wait two minutes If the message remains restart the computer Master DAQ Overflow Event rate too high in Instrument window Decrease the event rate or verify the threshold Too many analysis objects on worksheet or too many events displayed Delete analysis objects decr
49. bdbiosciences com Part No 640765 Rev A May 2006 BD FACSVantage SE Digital Option User s Guide BD Biosciences 2350 Qume Drive San Jose CA 95131 1807 USA Tel 877 232 8995 Fax 408 954 2347 Asia Pacific Tel 65 6 861 0633 Fax 65 6 860 1590 Europe Tel 32 53 720211 Fax 32 53 720452 Brazil Tel 55 11 5185 9995 Fax 55 11 5185 9895 Japan Nippon Becton Dickinson Company Ltd Tel 0120 8555 90 Canada Tel 888 259 0187 905 542 8028 Fax 905 542 9391 canada bd com Mexico Tel 52 55 5999 8296 Fax 52 55 5999 8288 2006 Becton Dickinson and Company All rights reserved No part of this publication may be reproduced transmitted transcribed stored in retrieval systems or translated into any language or computer language in any form or by any means electronic mechanical magnetic optical chemical manual or otherwise without prior written permission from BD Biosciences The information in this guide is subject to change without notice BD Biosciences reserves the right to change its products and services at any time to incorporate the latest technological developments Although this guide has been prepared with every precaution to ensure accuracy BD Biosciences assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information BD Biosciences welcomes customer input on corrections and suggestions for improvement BD
50. commended for pressures gt 45 psi 2 Install the tube holder For two way sorting see the following section e For four way sorting see Installing the Four Way Tube Holder on page 97 96 BD FACSVantage SE Digital Option User s Guide Installing the Two Way Sorting Hardware 1 Verify that the Fluidics Control knob is set to Off and that the deflection plates are not charged A A To prevent shock do not touch the deflection plates when the red warning light appears on the control panel 2 Install the adjustable support bracket below the center stream aspirator using the two metal thumbscrews 3 Install the pegs at the appropriate positions in the support bracket slide the collection tube holder onto the pegs e If you are using the 12 x 75 mm tube holder ports for cooling water face toward the front of the instrument e If you are using the 15 mL tube holder ports for cooling water face toward the rear of the instrument Installing the Four Way Tube Holder The four way tube holder provided with the digital option allows sorting into four tubes simultaneously Figure 4 11 The angle and height of the tubes can be adjusted to optimize sample collection during sorting The tube holder can accommodate any combination of Eppendorf 12 x 75 mm and 15 mL tubes Figure 4 11 Four way tube holder for 12x75 mm tubes Chapter 4 Sorting 97 NOTICE When used with 15 mL tubes the four way tube holder is designed to hold onl
51. concentration Figure 6 1 Calcium flux data 5 g 3 Lg N gt 2 Ze a T 25 x 1 000 130 BD FACSVantage SE Digital Option User s Guide Calcium Flux Optimization Before beginning this section do the following e Optimize the instrument electronics as described in Chapter 2 Instrument Setup and Optimization Ensure the appropriate filters are installed For the UV1 filter use 405 20 violet for the UV2 filter use 485 22 blue for the beam splitter use 505 SP See Appendix A Optical Configurations for the layout e Review the following section Using the Time Parameter Using the Time Parameter The Time parameter can be used to show how events change over time In calcium flux experiments the Time parameter is used to display the rate at which the cells in the sample respond to a stimulus The Time parameter is displayed on a fixed scale of 0 262 143 where each tick represents 10 ms Thus an event that appears at position 50 000 on the Time scale is equal to 8 min 20 sec an event that appears at 60 000 is equal to 10 minutes A plot can display up to 43 minutes of Time data When you append data to a recorded tube time is added to the existing data set Thus after appending 5 minutes of data to a 10 minute data set the Time parameter of the last event would appear at 90 000 NOTICE Do not restart data recording during a calcium flux experiment or data will be lost Chapter 6
52. d 150 000 2 Adjust the Sample Differential knob to decrease the event rate to approximately 200 events second 3 Alt click the current tube pointer to record data for the UV tube Alternatively click Record Data in the Acquisition Dashboard 4 Copy the third laser results into your QC log Copy the fluorescence channel means into the QC log and print out the worksheet for your records Figure 2 11 Figure 2 11 Results for third laser optimization 022306 UV 022306 UV UV 2A gt 1 000 110 200 250 1190 P 90004404 100 50 100 150 200 250 FSC A 1 000 022306 UV w 10 uv Count 100 200 300 N E Count 50 100 150 200 250 100 150 200 250 UV 1 A 1 000 UV 2 A x 1 000 Experiment Name Feb 2006 Specimen Name 022306 Tube Name uy Collection Date Feb 23 2006 1 25 49 PM UV1 A UV 1 A UV 2 A UV 2 A Population Mean cv Mean cv Kuv 121 265 8 6 116 840 8 7 DA Uv2 A 121 238 8 5 116 790 8 5 Chapter 2 Instrument Setup and Optimization 51 Reusing the Experiment To reuse this experiment do the following 1 Open the QC experiment 2 Right click the specimen and choose Duplicate without Data The three optimization tubes appear under the new specimen 3 Rename the specimen with today s date 4 Move the current tube pointer to the 488 nm tube The appropriate worksheet is shown in the Worksheet window 5 Proceed with optimizatio
53. d 150 000 2 Verify the FITC signal with the iris closed e Close the iris e Increase the Events to Display to 10 000 events e Create an interval gate on the FITC histogram peak e Click Acquire Data e After acquiring 10 000 events take note of the FITC signal ie print the worksheet or write down the FITC mean fluorescence for the peak e Decrease the Events to Display to 500 events 3 Open the iris Alt click the current tube pointer to begin recording data Alternatively click Record Data in the Acquisition Dashboard 4 After recording is complete draw an interval gate around each peak on the scatter and fluorescence histograms Adjust the gate on the FITC histogram if needed Mi Tip Double click the Autointerval Gate button and create all interval gates Then press Escape to unstick the button and readjust the gates as needed 5 Use the population hierarchy to rename each population defined by the interval gates To display the population hierarchy right click the plot and choose Show Population Hierarchy Select a population in the hierarchy and enter a new name to change it For example change P1 to FSC p1 P2 to FSC p2 and P3 to FITC See Figure 2 4 on page 38 Chapter 2 Instrument Setup and Optimization 37 6 Edit the statistics view to show only the named populations Right click the statistics view and choose Edit Statistics View Click the Populations tab and deselect the checkbox for the All Events
54. drive is on values in the Breakoff and Streams tabs are sent to the cytometer At startup the drop drive defaults to off ty 8 Drop Driveon Drop Drive off NOTICE The drop drive should be off when an experiment does not involve sorting e Test Sort generates test side streams based on the Drop sequence specified in the Breakoff tab when the button is clicked The drop drive must be on to enable the Test Sort button wy Test on Test Sort off Mi Tip Press the F12 key to switch Test Sort on and off e Attenuation decreases the amplitude of the drop drive oscillation when the button is clicked At startup attenuation defaults to off As a general rule turn on attenuation when sorting below 30 psi 3 M Attenuationon Attenuation off Chapter 4 Sorting 77 Using the Breakoff Controls Cells or particles flow through the BD FACSVantage SE nozzle tip in a fluid stream During sorting energy is applied to the stream to break it into droplets Droplets detach from the stream at the breakoff point typically a few millimeters downstream from the nozzle Drop drive controls in the Breakoff tab are used to adjust the breakoff point by changing the characteristics of the drop drive For examples of setting up the Breakoff tab for sorting see Adjusting Sort Settings on page 99 Streams Frequency F1 27 0 Yl Amplitude F2 20 0 st Phase F3 20 S Drop Delay F4 45 00 2t Drop Sequence h 0000200003000040000
55. e controlled by BD FACSDiva software When switched to Off the instrument is in analog mode and the electronics are controlled by BD FACStation software and the instrument control panel You can run the cytometer in either digital or analog mode but not both simultaneously although you can observe digital or analog processed data when operating in the alternate mode Digital Operation During operation in digital mode analog settings for gain threshold compensation and the event rate are displayed on the analog oscilloscope Because threshold and gain are controlled differently depending on the electronic mode these settings can differ from those displayed by the digital electronics For Chapter 1 Features of the Digital Option 17 18 an accurate event count monitor the Acquisition Dashboard in BD FACSDiva software rather than the analog oscilloscope When operating in digital mode the analog oscilloscope reflects adjustments made to the photomultiplier PMT voltages in BD FACSDiva software These adjustments will also change the data displayed in BD CellQuest or BD CellQuest Pro software However analog data is subject to any gains set in BD CellQuest software and thus might differ from the data displayed on the BD FACSDiva workstation For this reason it is important to use signals displayed on the analog oscilloscope and in BD CellQuest software for troubleshooting purposes only A Because digital data processing is d
56. ease the Display value or delete parameters from the Parameters tab 142 BD FACSVantage SE Digital Option User s Guide Electronics Troubleshooting continued Observation Possible Causes Recommended Solutions Instrument not Unknown Reset the digital electronics by responding in Status tab switching off the power switch on the back of the digital electronics module and then switching the power back on Restart the computer NOTICE If this occurs during sorting turn off the deflection plates before resetting the electronics Chapter 7 Troubleshooting 143 Acquisition Troubleshooting Observation No events in plots after clicking Acquire Data Possible Causes Current tube pointer not set to current tube Recommended Solutions Click to move the current tube pointer next to the appropriate tube Not in digital mode Switch the Digital control switch to On Viewing plots for a different tube Double click the current tube in the Browser to display the plots for that tube Incorrect population s in plot Right click the plot and choose Show Populations Verify that the appropriate populations are displayed Uncolored events in plot e Format the plot to display all events e Assign a color to the population displayed in the plot e Verify the population drawing order Current instrument configuration different from optical bench Verify that the current instr
57. ee Appendix A for suggestions Laser delay set incorrectly Adjust the laser delay settings See Optimizing Signals from the Second Laser Intercept on page 39 or Optimizing Signals from the Third Laser Intercept on page 46 Low area signal Area scaling too low Adjust area scaling for the corresponding laser Chapter 7 Troubleshooting 145 Acquisition Troubleshooting continued Observation Possible Causes Unexpected events in Incorrect logic in population plot hierarchy Recommended Solutions Verify the gating strategy Incorrect population s in plot Right click the plot and choose Show Populations Verify that the appropriate populations are displayed Incorrect drawing order Verify that the required population is not hidden by another population Right click the plot and choose Order Populations by Count Unexpectedly high event Threshold channel too low rate Adjust the threshold channel See Adjusting the Voltages and Threshold on page 59 Sample too concentrated Dilute the sample Event rate too high Decrease the event rate using the Sample Differential knob Air bubble Remove the air bubble Refer to the BD FACS Vantage SE User s Guide Test signal interference Turn off test signals from the instrument control panel Laser noise Put the instrument in Standby and adjust the FSC obscuration bar to remove the noise Decrease the
58. ell is selected In Initial mode only the Yield Mask is used at its maximum value thus recovery and yield are optimized at the expense of purity NOTICE Initial mode is equivalent to the Yield mode It is named differently as a reminder to use this as the initial mode when setting the drop delay using the Accudrop option In Fine Tune mode all masks are set to zero for deflecting the maximum number of drops This mode is used to fine tune the drop delay value using the Accudrop option Defining New Precision Modes Default Precision modes cannot be edited however you can create new modes and then choose them from the Precision Mode drop down menu 1 Choose Sort gt Sort Precision click Add Precision Mode Purity The current sort mode is duplicated and the YiedMask 32 Mask fields are enabled PurtyMas 32 Phase Mask 0 2 Optional Change the name of the mode in Single Celt ix the Precision Mode field aad cose 3 Enter values for the Yield Purity and Phase Masks Chapter 4 Sorting 93 4 Click to select the Single Cell checkbox if needed 5 Click Close The new mode is added to the Precision Mode drop down menu To delete a mode choose it from the drop down menu and then click Delete General Sorting Overview The following section presents a general overview of the main sorting adjustments For specific instructions see Setting Up for Sorting Into Test Tubes on page 96 or Setting
59. en wells on a plate or spots on a slide The Home location is used as the starting point at the Home location the far left stream should hit the center of the well in the top left corner of the sorting device Default Home location coordinates exist for each pre programmed collection device Before beginning a sort use the following procedure to verify the Home location and adjust it if needed 1 Perform sort setup Optimize the sort settings as if you were setting up for a two way sort at standard pressure see Adjusting Sort Settings on page 99 All steps are identical except for configuring the Streams tab when sorting into a plate or slide only the far left stream is used For step 5 on page 101 input the settings as shown in the following figure BA 12 psi 96 well xj Far Lett F5 a0 S d 2nd Drop F9 itl Left F6 v itl 3rd Drop F10 HET itl Right F7 o Yl 4th Drop F11 s Ed Far Right F8 o Yl 2 Install the collection device on the tray support 3 Choose Sort gt Home Device The Device Setup dialog box appears Figure 4 19 on page 112 If a Sort Layout is currently open the corresponding collection device will be selected in the list of devices Otherwise select the appropriate collection device in the list Chapter 4 Sorting 111 Figure 4 19 Setting the Home location Device Setup 6 Well Falcon Name 24 Well Costar Home Pos 1579 318 24 Well
60. eport Device 2 Tube Print Preview Report Date 2006 02 27 at 02 25 24 Print Report Export Mediurn Precision Yield Frequency 27 00 Yield Mask 32 Amplitude 20 00 Purity Mask 0 Phase 20 00 Phase Mask 0 Drop Delay 45 00 Single Cell Off Attenuation Off NOTICE After closing a Sort Layout all counter information is lost Therefore you should print a Sort Report immediately after sorting Chapter 4 Sorting 103 Calculating the Drop Delay Use the Accudrop option to determine the optimal drop delay setting for your sorting application For more information refer to the BD FACS Accudrop User s Guide Setting Up the Experiment The steps in this section show you how to set up an experiment for Accudrop optimization Because no data is recorded the experiment can be reused as often as you like 1 Create a new experiment and rename it Accudrop 2 Rename the first tube Accudrop Beads 3 With the Accudrop Beads tube selected in the Browser click on the Instr Settings gt Parameters tab in the Inspector and delete all parameters except FSC and SSC 4 Create an FSC histogram for the Accudrop Beads tube Defining the Bead Population Perform this procedure after adjusting the sort settings 1 Move the emission filter away from the camera 2 Inthe Sort Setup window click the button to turn on Test Sort YY 3 While viewing the streams on the Accudrop monitor adjust the micrometer dial to obtain even illumination
61. equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his or her own expense Shielded cables must be used with this unit to ensure compliance with the Class A FCC limits This Class A digital apparatus meets all requirements of the Canadian Interference Causing Equipment Regulations Cet appareil num rique de la classe A respecte toutes les exigences du R glement sur the mat riel brouilleur du Canada History Revision Date Change Made 341756 Rev A 330798 Rev A 330802 Rev A 334555 Rev A 338648 Rev A 640765 Rev A 8 01 1 02 05 02 11 02 10 04 05 06 Production release for software version 1 0 Updated for software version 2 0 enhanced performance database redesign and data management utility scalable data display instrument settings features Next button more copy paste ability plot display features Refer to the ReadMe file for details Updated for software version 2 1 enhanced performance workspace redesign with separable components Browser level folders functioning Acquisition pointer Sort Layout redesign objects duplicated by dragging drill down gating log decade gridlines on plots view hide gate boundaries context sensitive cursors histogram smoothing gate changes downloaded during sorting automatic acquisition during record sort experiment import export Ratio Scaling factor per rat
62. er nozzle opening requires a higher frequency at a given sheath pressure The standard nozzle holder is not intended to be used at sheath pressures gt 20 psi For higher sorting pressures verify that the high speed sort head provided with the BD TurboSort Plus option is installed 4 Adjust the amplitude to optimize the breakoff point minimize stream noise Vv Tip Breakoff values can be adjusted using the keyboard Press the indicated function key ie press F2 for amplitude and use the up and down arrow keys to adjust the value Hold the Control key down while pressing the arrow keys to adjust the values in larger increments 100 BD FACSVantage SE Digital Option User s Guide 5 6 Input approximate streams settings based upon the sort direction Click the Streams tab in the Sort Setup window to input settings Figure 4 14 If you recalled settings for the Breakoff tab verify that the Streams tab is set appropriately Figure 4 14 Preliminary Streams settings for a two way upper and four way lower sort BA 11 psi 2 way x BreakOtt Far Lett F5 oo St 2nd Drop F9 tl Letrey solt 3rd Drop Ft0 gt 10 St Right F7 40 St 4th Drop F11 os Stl Far Right F8 oo Stl two way sort 11 psi BreakOtt Far Left F5 a0 St 2nd Drop F9 tl Letrey solt 3rdDrop Ft0 10t Right F7 ao EL 4th Drop F11 os Stl Far Right F8 a Eu four way sort Adjus
63. er sorting A Sort Report contains the following Header information tube name Sort Layout name type of collection device and the date and time of printing e Sort settings sort setup values information in the Breakoff and Streams tabs precision mode and masks definition Acquisition counters threshold count processed events count electronic conflicts count and elapsed time Chapter 4 Sorting 87 e Sort counters counter values per sort destination or total sort count if sorting sequentially e Sort Layout population s sort count and target event count for each sort location field The Sort Report window contains a File menu where you can choose to print or export the report Exported comma separated values CSV files can be opened with a spreadsheet application such as Microsoft Excel Sort Report File Page Setup Sort Report Device 2 Tube Print Preview Report Date 2006 02 27 at 02 25 24 Print Report Export Medium Precision Yield Frequency 27 00 Yield Mask 32 Amplitude 20 00 Purity Mask 0 Phase 20 00 Phase Mask 0 Drop Delay 45 00 Single Cell Off Attenuation Off Conflict Resolution with BD FACSDiva Software During sorting the cytometer deflects drops based on the characteristics of the particles in each drop and where the user wants to deflect them Drops are deflected depending on the type of target particle where the particle is contained in the drop or whether the drop
64. er switch and start up the BD FACSDiva workstation start up the BD FACStation computer if needed After logging on to Windows launch BD FACSDiva software by double clicking the shortcut on the desktop Verify that the instrument is connected by checking the Instrument window in the software The message Instrument Connected appears after the cytometer connects to the workstation this can take several minutes If the Instrument Disconnected message remains refer to the troubleshooting suggestions in the software manual Switch on the main pressure toggle switch Remove the sample tube from the sample injection port SIP Turn the Fluidics Control knob to Fill for 10 20 seconds to remove air bubbles Turn the Fluidics Control knob to Run Chapter 2 Instrument Setup and Optimization 25 Instrument Optimization and Quality Control 26 Instrument optimization is a process that ensures consistent instrument performance on a daily basis given the same laser power alignment sample and instrument settings Daily instrument optimization consists of the following copying instrument settings from a previous similar setup running an alignment sample beads or prepared cells optimizing signals from the laser beams When instrument settings and the alignment sample are kept constant changes in the means and CVs indicate variations in instrument performance over time Keep track of means and CVs in a quality control QC
65. ered in this chapter Criteria for DNA Experiments on page 116 CEN Optimization on page 117 e CTN Resolution on page 124 Optimization for Data Recording on page 127 115 Criteria for DNA Experiments 116 In DNA experiments the flow cytometer must provide the following e resolution e linearity e ability to distinguish singlets from aggregates Obtaining good resolution for the DNA signal depends on proper sample preparation and instrument optimization of the optics and fluidics The resolution of a flow cytometer can be assessed by measuring the CV of a reference particle the lower the CV the better the resolution Linearity is critical for DNA experiments To verify the linearity of DNA data the pulse area signal is used to measure the amount of DNA fluorescence detected from cells and nuclei For example the G M peak should be located at twice the mean channel of the Go G peak Figure 5 1 Figure 5 1 Area signal measures amount of DNA fluorescence DNA QC Kit CEN E P ets 150 200 250 LA Doublet discrimination or the ability to resolve singlets from aggregates is also important for DNA experiments Aggregated cells or nuclei are detected as events that have two or more times the amount of singlet fluorescence For cell cycle analysis it is important to resolve singlets from aggregates because doublets of Go G cells have the same amount of DNA fluorescence as singlet G2 M cells Therefore these doub
66. es e details of recent instrument performance For instrument support from within the US call 877 232 8995 For support from within Canada call 888 259 0187 Customers outside the US and Canada contact your local BD representative or distributor Limitations For Research Use Only Not for use in diagnostic or therapeutic procedures About This Guide xi xii BD FACSVantage SE Digital Option User s Guide Features of the Digital Option The following topics are covered in this chapter e About the Digital Option on page 14 Components on page 15 13 About the Digital Option 14 The digital option adds digital capability to the current analog functionality of the BD FACSVantage SE flow cytometer Digital electronics continuously digitize the cytometer s signals after linear amplification Logarithmic conversion display is achieved using lookup tables Continuous digitization eliminates dead time improving sort yield and facilitating better sort decisions On the BD FACSVantage SE flow cytometer the digital option can process up to eight fluorescence and two scatter channels with full interbeam compensation between both height and area parameters from any laser BD FACSDiva software can be programmed to sort a specified number of particles from multiple gates into a variety of sorting devices including tubes plates and slides The new four way tube holder enables sorting into four tubes simultaneously
67. es can be defined see Creating a Custom Device on page 113 Sort Report displays a report showing the sort settings acquisition counters and Sort Layout information from the current sort See Sort Report on page 87 Sort Setup Controls The Sort Setup window contains controls used to set up the instrument for sorting Display the Sort Setup window by clicking the Sorting button in the Workspace toolbar l FA Medium x Frequency F1 27 0 St Amplitude F2 20 0 YH a Phase F3 20 St Drop Delay F4 45 00 2i Drop Sequence 10000200003000040000 a H Z Drop Drive Test Sort Attenuation sort setup buttons The Sort Setup window contains drop drive controls in the Breakoff tab and stream charging controls in the Streams tab The values entered in each tab apply Chapter 4 Sorting 75 globally to BD FACSDiva software and are not saved with experiments or tubes The default values at startup are the last set of values used by the software Sort setup values for different applications can Sort be saved and recalled using the Sort Setup Sort Setup Save As command in the Sort menu Default settings Sort Precision Delete are provided for high medium and low Recall pressure sorts additional custom settings can be defined and saved See Saving and Recalling Home Device Custom Devices Sort Setup Values on page 80 Adjusting Settings Within
68. ext Tube in the Acquisition Dashboard change amp jn the name of the new tube to CTN Instr Settings 4 DNA QC Kit cen Clicking Next Tube duplicates the CEN tube and analysis L cm objects The new plots and statistics view appear below the previous objects on the worksheet Acquisition starts automatically and events appear in the plots Notice the lack of resolution between the singlets and doublets Figure 5 7 Figure 5 7 Unresolved singlets and doublets in unzoomed plot 150 200 250 PLA x 1 000 124 BD FACSVantage SE Digital Option User s Guide 4 Adjust the PI voltage to place the first peak at approximately 50 x 103 on the PI A axis 5 Focus the laser using the Excitation Beam Focus wheel As the laser is focused the area measurement will decrease Figure 5 8 Figure 5 8 Decrease in area measurement as laser is focused DNA QC Kit CTN DNA QC Kit CTN 50 100 150 200 250 150 200 250 PLA x 1 000 PLA x 1 000 6 Adjust area scaling Click the Laser tab in the Instrument window Adjust area scaling for the first laser until the PI A intensity is similar to the PI H intensity Figure 5 9 Area scaling adjusted DNA QC Kit CTN 150 200 250 PLA x 1 000 7 Adjust the PI voltage to place the singlets at approximately 50 x 103 if necessary Chapter 5 DNA Analysis 125 8 Optional Use the Zoom In button to magnify the area showing the singlets and doublets on the PI A vs PI W plot Ma
69. f necessary adjust the Alpha and Theta controls to correctly position the fluid stream Install the alignment sample onto the cytometer turn the Fluidics Control knob to Run Set the sample differential pressure to its standard level for this alignment procedure Verify that the current tube pointer is in front of the 488 nm tube in the Browser click once on the pointer to begin acquisition Alternatively click the Acquire Data button in the Acquisition Dashboard events appear in the plots Chapter 2 Instrument Setup and Optimization 33 34 NOTICE During digital operation use the analog pulses displayed on the analog oscilloscope for troubleshooting purposes only If necessary adjust the threshold gains and log settings in BD CellQuest or BD CellQuest Pro software to change the analog pulse display These will not affect digital data but can help in alignment 6 Maximize the FSC signal Adjust the Excitation Beam Focus wheel and Y control to obtain the highest FSC signal intensity If the FSC signal is out of standard range check the position of the FSC obscuration bar 7 Maximize the FITC signal Adjust the X control the Fluorescence Focus control knob and the Fluorescence channel height adjustment wheel to obtain the highest FITC signal intensity Vv Tip If you don t see any signal increase the PMT voltage or turn on Log before adjusting the stream controls 8 Close the FL1 iris incrementally as you continue op
70. gG PE Mouse IgG PerCP Cy5 5 CD8 APC Mouse IgG APC Cy7 Mouse IgG FITC Mouse IgG PE Mouse IgG PerCP Cy5 5 Mouse IgG APC CD8 APC Cy7 Chapter 3 Running Samples 5S5 Creating the Experiment Before you begin optimizing settings it is important to verify the instrument configuration and create an experiment containing appropriate parameters for the assay 1 Choose Instrument gt Instrument Configuration and verify the current configuration Make sure the configuration lists the parameters to be measured and that the channels correspond to the optical bench configuration For this example the instrument configuration should include the following parameters FITC PE PerCP Cy5 5 APC and APC Cy7 A For accurate data results the instrument optics must match the current instrument configuration 2 Click the corresponding buttons in the Workspace toolbar to display the Browser Instrument 1 Inspector P Worksheet and Acquisition Dashboard J windows as needed 3 Optional Create a folder for your experiment Select the icon for your database and press Ctrl N rename the folder appropriately Refer to the BD FACSDiva Software Reference Manual for ideas on how to organize experiments M Tip To place an experiment inside a folder select the folder before you create the experiment 4 Click the corresponding button s to create an experiment specimen and tube rename the experiment with an appropria
71. gnify the area from the left of the singlets to the right of the doublets including only the area of interest The zoomed in area should be long and narrow Figure 5 10 Doublet discrimination in zoomed in plot 1 000 190 10 170 a 150 a Paes 4 50 60 70 80 90 100 110 120 PLA x 1 000 9 Optional Draw a gate around the singlet CTN 10 Decrease the event rate to approximately 200 events second 11 Click Record Data to save the data print the worksheet 12 Remove the CTN tube from the cytometer put the instrument in Standby 126 BD FACSVantage SE Digital Option User s Guide Optimization for Data Recording Optimize the instrument settings for the actual sample 1 Install the sample tube and optimize the FSC and SSC signals 2 Optimize the PI voltage to place the singlets close to channel 50 x 10 3 Verify the doublet discrimination by zooming in on the PI A vs PI W plot If the singlets are not resolved repeat step 6 on page 121 After optimizing the instrument settings record data for each sample tube Export data files for analysis in a third party application such as ModFit LT Chapter 5 DNA Analysis 127 128 BD FACSVantage SE Digital Option User s Guide 6 Calcium Flux The following topics are covered in this chapter Intracellular Calcium Concentration on page 130 Calcium Flux Optimization on page 131 Measuring Calcium Flux on page 137 129 Intracellular Calcium C
72. hat the unstimulated sample is still running adjust the Threshold Rate to approximately 200 events second The Threshold Rate is displayed in the Acquisition Dashboard 3 Click Record Data A Do not stop recording as you perform the following steps If recording is stopped append subsequent data to the unstimulated sample data 4 When approximately 10 000 events have been recorded turn the Fluidics Control knob to Standby and remove the unstimulated sample tube from the cytometer 5 Add the stimulus to the tube and mix thoroughly 6 Reinstall the tube on the cytometer turn the Fluidics Control knob to Run After a few seconds the Cat concentration begins to increase on the time vs ratio plot Figure 6 4 Figure 6 4 Cellular response to stimulus over time CA1 P1 Ratio UV1 A UV2 a 1 000 7 T PPP 1p 5 25 i Time x 1 000 L unstimulated stimulus sample added Chapter 6 Calcium Flux 137 Acquisition and recording stop automatically when the Stopping Time value is reached 7 After acquisition stops remove the tube from the cytometer 8 Clean the fluidics system with 10 bleach for 5 minutes and then with deionized water for 5 minutes NOTICE Make sure to remove any remaining stimulus that would activate cells in subsequent samples 9 Torun another sample install the next tube onto the cytometer 10 Click Next Tube rename the new tube as appropriate 11 Repeat steps
73. he following adjustments e Adjust the FSC and SSC voltages to place the sample on scale in the FSC vs SSC dot plot e Adjust the FSC threshold to remove debris without cutting into the population of interest Draw a gate around the lymphocytes use the population hierarchy view to rename the population Lymphocytes Figure 6 3 Adjusting FSC and SSC voltages Ca 1 All Events Chapter 6 Calcium Flux 135 5 Format the remaining two dot plots to show the Lymphocyte population Select the two plots right click inside one of the plots and choose Show Populations gt Lymphocytes 6 Adjust the UV1 and UV2 voltages to optimize the signal The signal should extend along the UV2 axis and should be slightly off the baseline for both axes 100 150 200 250 UV 1 Violet A amp 1 000 7 Adjust the ratio scaling to set the baseline between 0 50 000 Select the Ca 1 tube in the Browser and click the Instr Settings gt Ratio tab in the Inspector To adjust the setting select the value in the Scaling field enter a new value and press Enter Repeat as needed to achieve the required results 100 150 200 260 Ratio UV 1 AsUyoos 00 5 A n q 48 a 5 f f 7 7 T f C 7 T T 50 100 150 200 Time x 1 000 136 BD FACSVantage SE Digital Option User s Guide Measuring Calcium Flux Do the following to record data for a calcium flux experiment 1 Change the Events to Display to 50 000 events 2 Verify t
74. ical bench configuration For accurate data results the instrument optics must match the current instrument configuration Click the corresponding buttons in the Workspace toolbar to display the Browser 9 Instrument 1 Inspector Worksheet and Acquisition Dashboard d windows as needed Optional Create a folder for instrument New QC Folder Gi Gi P 2 g Fi Hi E In the Browser select the icon for your database icon Administrator Folder _00 a gt Shared View database and click the New Folder button in the Browser toolbar Rename the folder with your name Alternatively name the folder Instrument OC or create an Instrument QC folder inside another folder Refer to the BD FACSDiva Software Reference Manual for ideas on how to organize experiments M Tip To place an experiment inside a folder select the folder before you create the experiment Chapter 2 Instrument Setup and Optimization 27 4 Click the corresponding button s to create an experiment specimen and tube rename the experiment with an appropriate name For example use the current month and year Instrument Opt or the operator s initials followed by an appropriate identifier 5 Rename the new specimen with today s date rename the first tube 488 nm This tube will be used to optimize signals from the first laser 6 Click to set the current tube pointer next to the 488 nm tube Your experiment should loo
75. icles kit 117 setting up experiment 118 verifying linearity 116 doublet discrimination 116 122 126 drop charge 79 conflicts 88 correction factor 79 delay 78 104 drive 77 sequence 78 starting values 102 E editing Sort Layouts 85 eight color configuration 157 158 electronics aborts 149 about 14 control switch 17 troubleshooting 142 emptying waste 24 error messages instrument disconnected 142 instrument not responding 143 Master DAQ overflow 142 Unable to move stage 151 upgrading firmware 142 events not showing in plots 144 146 rate troubleshooting 146 147 troubleshooting 149 Excitation Beam Focus wheel 32 Index 163 experiments AccuDrop optimization 104 calcium flux 132 CEN optimization 118 immunophenotyping 64 instrument optimization 27 reusing 52 sample optimization 56 exporting Sort Reports 88 F FACStation 20 Far Left stream 79 Far Right stream 79 Fine Tune mode 93 fluorescence channel height adjustment wheel 32 focus control knob 32 signal troubleshooting 145 four way sorting about 19 installing hardware 97 tube placement 98 frequency about 78 optimal ranges 100 function keys troubleshooting 76 150 G gating data 67 global worksheets creating 64 previewing data 63 70 viewing 70 H hardware biohazardous 94 four way sort installing 97 plate sorting installing 110 two way sorting installing 97 Home Device 75 Home location adjusting 75 111 custom devices 113 immunophenot
76. ifferent from analog data processing do not save BD CellQuest files collected while operating in digital mode NOTICE In digital mode most controls on the instrument control panel are inactive see Figure 1 4 Equivalent controls can now be found in BD FACSDiva software Figure 1 4 Instrument controls shaded active in digital mode DROP DRIVE CONTROLS Co CONTRAST J O BRIGHT A A O V V H HOLD STREAM CONTROLS O deflection plates on off center stream control To p C4 drop strobe on off F stream lamps on off VIEWING SORT CONTROLS MARK 70 A A plate voltage control e Vv Vv NR Vec MIN MAX MIN MAX MIN MAX Q OFF Q OFF DROP 3 PLATE DROP 2 DROP DRIVE INDEX CHARGE CHARGE VOLTAGE ATTENUATION SORT BD FACS Vantage SE Digital Option User s Guide Analog Operation During operation in analog mode instrument controls on the BD FACSVantage SE control panel are enabled Any changes made within BD FACSDiva software will not be registered on the analog oscilloscope nor in BD CellQuest software However changes made in BD FACSDiva software will be reflected on the digital oscilloscope Four Way Sorting Tube Holder The four way tube holder provided with the option allows sorting into four tubes simultaneously Figure 1 5 The angle and height of the tubes can be adjusted to optimize sample collection during sorting Figu
77. in material damage data A loss minor or severe injury or death A Electrical danger A Laser radiation Biological risk a Although these symbols appear in color on the instrument they are in black and white throughout this user s guide their meaning remains unchanged Table 2 Text and keyboard conventions Convention Use MV Tip Highlights features or hints that can save time and prevent difficulties NOTICE Describes important features or instructions Italics Italics are used to highlight book titles and new or unfamiliar terms on their first appearance in the text gt The arrow indicates a menu choice For example choose File gt Print means to choose Print from the File menu Ctrl X When used with key names a dash means to press two keys simultaneously For example Ctrl P means to hold down the Control key while pressing the letter p x BD FACSVantage SE Digital Option User s Guide Technical Assistance For technical questions or assistance in solving a problem Read the section of the user s guide specific to the operation you are performing e See Chapter 7 Troubleshooting If additional assistance is required contact your local BD Biosciences technical support representative or supplier When contacting BD Biosciences have the following information available product name part number and serial number software version and computer system specifications any error messag
78. io Area Scaling factor per laser Refer to the ReadMe file for details Instrument features and operation separated from general software information Instrument procedures updated to reflect version 2 2 of the software Refer to the BD FACSDiva Software User s Guide for more information Changed product name and book title to BD Digital Electronics Option Updated software terminology and screen shots for BD FACSDiva software version 4 1 Updated software terminology and screen shots for BD FACSDiva software version 5 0 changed book title Contents About This Guide Conventions Technical Assistance Limitations Chapter 1 Features of the Digital Option About the Digital Option Components Digital Electronics Module Qc ce cec cec cee Four Way Sorting Tube Holder Q Q BD FACSDiva Workstation Q Q coec ence eens Chapter 2 Instrument Setup and Optimization Starting Up the Instrument cece teen eens Instrument Optimization and Quality Control c Preparing the Alignment Sample 0 00 c eee eee eee eens Setting Up the Experiment occ cece eee eens Optimizing Signals from the Primary Laser Optimizing Signals from the Second Laser Intercept Optimizing Signals from the Third Laser Intercept Reusing the E
79. is set to the maximum to obtain the greatest number of particles because the Purity Mask is also at the maximum only drops with a target particle will be sorted Sorting in Purity mode results in a sorted sample that is highly pure at the expense of recovery and yield In 4 Way Purity mode the Purity Mask is set to the maximum so only drops free of contaminating particles will be sorted The Yield Mask is set to zero to ensure that residual charges from adjoining drops do not degrade the quality of side streams The 4 way Purity mode is recommended for four way sorting where precise deflection is required 92 BD FACSVantage SE Digital Option User s Guide In Yield mode only the Yield Mask is used at its maximum value thus recovery and yield are optimized at the expense of purity In Single Cell mode the Purity Mask is set to the maximum so only drops containing a target particle will be sorted The Phase Mask is set at half the maximum so only particles centered within the sorted drop are deflected Drop trajectory and count accuracy are optimized at the expense of yield This mode is recommended for single cell sorting or situations where precise counting is required Vv Tip Select the Single Cell checkbox to obtain the highest quality side streams and the most accurate counts When the checkbox is selected drops containing two target events acceptable with a Purity Mask are discarded The Yield Mask is disabled when Single C
80. ition controls e BD FACSDiva gating and statistics buttons Review the corresponding chapters in the BD FACSDiva Software Reference Manual if needed Starting Up the Instrument Follow these instructions to start up the instrument for operation in digital mode 1 Turn on the laser water 2 Turn on the laser s In general allow the lasers to warm up for at least 30 minutes Refer to the BD FACSVantage SE User s Guide for specifications for each laser 3 Open the vacuum source and air supply 4 Verify that the sheath container is full and the waste container is empty If needed empty the waste AA All biological specimens and materials coming into contact with them can transmit potentially fatal disease To prevent exposure to biohazardous agents expose waste container contents to bleach 10 of total volume before disposal Dispose of waste in accordance with local regulations Use proper precautions and wear suitable protective clothing eyewear and gloves 24 BD FACSVantage SE Digital Option User s Guide 11 Before proceeding with step 5 verify that the four way tube holder is not installed If you turn on the power with the holder installed sorting hardware for the BD CloneCyt Plus option could catch on it potentially damaging the motor Turn on the cytometer main power switch Switch on the digital control switch if necessary The switch must be On to operate in digital mode Turn on the computer main pow
81. k similar to the following Feb 2006 3 Instr Settings Global Worksheets current tube 022306 pointer J5 N Edit the experiment level instrument settings M Tip Save space in the database by collecting data for only those parameters that will be used in the experiment e In the Instrument window select the Parameters tab and delete any unnecessary parameters e Deselect the Log checkboxes for FITC and PE When aligning with beads you might also need to deselect the checkbox for PerCP Cy5 5 Instrument x Status Parameters Threshold Compensation Ratio Laser Parameter Voltage Log PE 1 1 lt 1 a a PerCP Cy5 5 28 BD FACSVantage SE Digital Option User s Guide 8 In the Acquisition Dashboard set the Events to Record to 10 000 events and the Events to Display to 500 events EFE Acquisition Dashboard x Current Activity Active Tube Well Threshold Rate Stopping Gate Events Elapsed Time 488 nm 0 evtis 0 evt 00 00 00 Basic Controls gt V Next Tube E Acquire Data OB Record Data Acquisition Setup Storage Gate M Ai Everts v Events To Record 10000 evt v Stopping Time sec o St Stopping Gate RN ai Events v Events To Display 500 evt x M Tip Decreasing the number of displayed events will increase the data refresh rate 9 Create the following plots on the global worksheet e FSC vs SSC and FSC vs FITC dot p
82. knob Fluorescence channel height adjustment wheel and FL1 FL2 beam splitter as necessary NOTICE PI stained nuclei are used in this exercise so the fluorescence signal will be generated by the first laser and collected in the FL2 channel If you are setting up for an experiment using a different laser and detector channel peak the signals from that laser using the appropriate rotators translators and beam splitters 3 Decrease the event rate to approximately 200 events second 4 Open the FL1 FL2 iris and check the CV of the Singlet population e If the CV is within an acceptable range go to step 10 on page 122 e Ifthe CV is not within an acceptable range repeat steps 1 through 4 in this section Chapter 5 DNA Analysis 123 CTN Resolution Singlets can be distinguished from aggregates based on size With BD FACSDiva software aggregates can be resolved from singlets on an area vs height plot in conjunction with an area vs width plot On the area vs height plot singlets can be distinguished from doublets by their height on the y axis singlets have slightly more height On the area vs width plot singlets are distinguished from doublets by the width measurement singlets have a smaller width measurement Discriminating singlets from aggregates enhances the accuracy of cell cycle analysis Running CTN 1 2 Install the CTN sample tube onto the cytometer Adjust the event rate to approximately 500 events second Click N
83. late voltage contol O 7 7 4 r MIN MAX MIN MAX MIN MAX Q OFF Q OFF INDEX DROP 2 DROP 3 PLATE DROP DRIVE CHARGE CHARGE VOLTAGE ATTENUATION SORT Drop drive and side stream controls in the Sort Setup window See Sort Setup Controls on page 75 Sorting instructions controls and counters in the Sort Layout window See Sort Layout on page 81 Sort Precision modes Sort Layouts and sorting device commands in the Sort menu Choose commands in the Sort menu for the following sert Sort Setup b Sort Precision Sort Precision opens a dialog box where you can h d fi S P TE d f h dli Ea New Sort Layout choose or define a Sort Precision mode for handling open sort tayout sorting conflicts see Conflict Resolution with Tome Deda BD FACSDiva Software on page 88 Custom Devices Sort Report BD FACSVantage SE Digital Option User s Guide New Sort Layout opens the default 2 Tube Sort Layout where other sorting instructions can be specified see Sort Layout on page 81 Open Sort Layout opens an existing Sort Layout A Sort Layout must be selected in the Browser for this menu command to be enabled Alternatively double click any Sort Layout to open it Home Device opens a dialog box containing commands to move the tray support arm either manually or to the home position see Adjusting the Home Location on page 111 Custom Devices opens a dialog box where custom devic
84. lect a row or column header to select all fields in that row or column After adding a population it will be written to all selected fields at once Specify whether to save sort conflicts by selecting the Save Conflicts checkbox This checkbox is enabled only when using a two or four tube layout When selected all sort conflicts are sorted into a default location e Fora two tube layout conflicts are sorted to the right no other populations can be sorted to the right most tube za Sort Sample Sort Layout xj Device Precision Target Events Save Conflicts 2 Tube w IPurity v Vv Left Conflicts Continuous NT For a four tube layout conflicts for the Far Left tube are sorted to the left conflicts for the Far Right tube are sorted to the right No other populations can be sorted into the center most tubes z Sort Sample Sort Layout x Device Precision Target Events Save Conflicts 4 Tube Y Purity bg z a v Far Right Lett Conflicts Continuo Right Conflicts Continu 84 BD FACSVantage SE Digital Option User s Guide Editing a Sort Layout To change the number of events for any population center a new number in the Target Events field To remove a population from a sort location field select the field and then choose the corresponding population from the Delete menu To clear all populations from a field select the field and then choose Clear All Using Sor
85. lets accumulate in the same fluorescence area channel as singlet G M cells Figure 5 2 on page 117 BD FACSVantage SE Digital Option User s Guide Figure 5 2 Doublet discrimination 8 a x x a 150 200 250 1 000 NOTICE Before beginning this chapter do the following e Prepare biological standards for instrument quality control using the BD DNA QC Particles kit Catalog No 349523 Prepare one tube each of chicken erythrocyte nuclei CEN and calf thymocyte nuclei CTN sample according to the kit instructions The CEN sample is used for instrument optimization and to check instrument resolution CV and linearity The CTN sample is used to verify the system s ability to resolve singlets from aggregates e Optimize the instrument electronics as described in Chapter 2 CEN Optimization Use the following procedure to set up BD FACSDiva software for a DNA experiment that uses propidium iodide PI as the DNA staining dye eg the BD DNA QC Particles kit If you are using another sample type modify the steps accordingly Chapter 5 DNA Analysis 117 Setting Up the Experiment 1 Choose Instrument gt Instrument Configuration and verify the current configuration Make sure that the current configuration lists the PI parameter and that the channels correspond to the optical bench configuration The PI parameter should be assigned to Laser 1 and Channel FL2 A For accurate data results the i
86. llowing plots on the global worksheet e FSC vs SSC e CD3 PerCP Cy5 5 vs CD16 56 PE e CD3 PerCP Cy5 5 vs CD19 APC e CD3 PerCP Cy5 5 vs CD8 APC Cy7 e CD3 PerCP Cy5 5 vs CD4 FITC 3 Create a population hierarchy and a statistics view and move them below the plots on the worksheet 4 Draw a gate around the lymphocytes use the population hierarchy to rename the population Lymphocytes 5 Select all plots except the FSC vs SSC plot and specify to show only the Lymphocyte population Chapter 3 Running Samples 67 68 10 11 12 13 Select all plots and click the Title tab in the Plot Inspector select the checkboxes to display the tube and population names in the plot titles Inspector x Plot Title Labels Dot Piot Title Content I Custom Title Title Font Size 12 Color Face SansSerit K V taic l Bold Edit the statistics view to show only the Lymphocyte population and to display the mean for all fluorochromes Draw a region around the CD3 positive population on the CD3 PerCP Cy5 5 vs CD16 56 PE plot name the population T Cells Draw a region around the CD16 56 positive population on the same plot name the population NK Cells Draw a region around the CD19 population on the CD3 PerCP Cy5 5 vs CD19 APC plot name the population B Cells Select the T cell population in the population hierarchy view draw a region around the double positive population on the CD3 PerCP Cy5 5 vs C
87. lor Configuration amp l Current Configuration 6 color alternate Configurations Parameters Configuration Modified Date Parameter Laser Detector 6 color 4 3 06 9 44 00 AM FSC 1 Fse 7 color 4 13 06 9 44 00 AM SSC 1 ssc 8 color 1306 9 44 00 AM FITC 1 FLA 6 color alternate 4 3 06 3 09 45 PM PE 1 FL2 PE Cy7 1 FL3 uvi 3 FL4 UV2 3 FLS APC Cy7 2 FL6 APC 2 FL7 PE Cy5 J FL8 Add Set Duplicate Import Export OK Cancel FITC FL1 530 30 575 26 S pT PE FL2 Gt 560 SP PE Cy5 FL8 710 20 S 740 LP 750 LP or 780 60 C eto PID Q PE Cy7 FL3 405 20 780 60 UV1 FLA D Q APC Cy7 FL6 505 SP 710 LP 485 22 S S 660 20 SSC amp UV2 FL5 EO Appendix A Optical Configurations APC FL7 157 Alternate Eight Color Configuration Configuration gt Current Configuration 8 color alternate Configurations Parameters Configuration Modified Date Parameter Laser Detector 6 color 413 06 9 44 00 AM IFSC d FSC 7 color 4 13 06 9 44 00 AM ssc J SSC 8 color 413 06 9 44 00 AM FITC id FLA 6 color alternate 4 13 06 3 09 45 PM PE d FL2 2 PI PE Cy A FL3 Hoechst Blue B FL4 Hoechst
88. lots e FSC FITC PE and PerCP Cy5S 5 histograms M Tip To easily create multiple plots double click a plot button to keep the button selected Click multiple times within the worksheet to create the required number of plots When you are finished click any other button to undo the selection 10 Resize the plots so that they fill 2 3 of the worksheet M Tip To resize multiple plots simultaneously resize one plot and then press Ctrl A to select all other plots Click the Resize button to make all plots the same size as the first plot 11 Right click any plot on the worksheet and choose Create Statistics View Chapter 2 Instrument Setup and Optimization 29 12 Edit the statistics view as follows e Right click the statistics view and choose Edit Statistics View e Select the Populations tab and deselect the checkboxes for Events and Parent All Events Decimal Places e Set up the Statistics tab to display the mean and CV for FSC and each fluorescence channel detected by the 488 nm laser Edit Statistics View Header Populations L C C r r r Al cv Max Min SD Mode FSC A Oo C O Vv R r r mj SSC A r C r C r D FITC A C r C Vv R GB r r m PE A C C C R R m d O m PerCP Cy5 5 A r E r Vv Vv G Oo m UV1 A r O C C C C m m mj UV2 A r r r mj C C C r C APC A r r C a B mj d dO Time C m C m C C C C Decimal Places 0 0 0 1 0 0 0 0
89. n Mi Tip You can also save the specimen as a panel template Refer to the BD FACSDiva Software Reference Manual or the software online help for instructions 52 BD FACSVantage SE Digital Option User s Guide 3 Running Samples This chapter describes how to use BD FACSDiva software to record and analyze sample data If this is your first time using BD FACSDiva software BD recommends that you first practice the steps in this chapter using BD Calibrite beads This exercise will familiarize you with digital data and help you establish target channel values The following topics are covered in this chapter e Optimizing Settings Using Instrument Setup on page 54 Recording and Analyzing Data on page 63 53 Before Beginning This Chapter Before running samples start up the instrument and optimize the electronics as described in your instrument manual To perform the steps in this chapter you should be familiar with the following e General instrument operation Refer to the BD FACS Vantage SE User s Guide if needed e General software components workspace components instrument and acquisition controls buttons for data analysis Review the corresponding sections of the BD FACSDiva Software Reference Manual Optimizing Settings Using Instrument Setup 54 Before you record data for a sample instrument settings should be optimized to position the cells of interest on scale for scatter and fluorescence parameter
90. n eens 96 Setting Up for Sorting Into Test Tubes 02 cece eee eee eee eee 96 Installing the Sorting Hardware eee ee eens 96 Adjusting Sort Settings 20 0 0c ccc cece ence teens 99 Calculating the Drop Delay Q Q cc eee eens 104 Setting Up the Experiment 0 0c cece eee ences 104 Defining the Bead Population Qo cc ccc ccc 104 Sorting Beads to Determine the Drop Delay 106 vi BD FACSVantage SE Digital Option User s Guide SOLtING aS RE aS Pb ae SAD OES ied TPO Sade PO ae BS 107 Setting Up the Experiment Qo coec eke ecce 108 Starting and Monitoring the Sort Qa cc occ eee 109 Setting Up for Sorting Into a Plate or Slide Q Q 109 Installing the Sorting Hardware 00 cece cece eee eens 110 Adjusting the Home Location acoc ceca 111 Creating a Custom Device 00 cece ee eee ene eens 113 Chapter 5 DNA Analysis 115 Criteria for DNA Experiments oo cece teen eens 116 CEN Optimization 4 6 45 4 404544 R A USS Aa VA R 4 NA S 117 Setting Up the Experiment acc cece eee eens 118 Running CEN c n 2252242 b L 40 28a R R tet ee a akon 120 CTN Resol tion s 4 2 nen 04490 CEA Dt 4 R GAR RA R VR nes be 124 R nning CIN issu sade ha ead edie Deedee Red 4A S A0 474 4 68 124 Optimization for Data Recording ccc ec cec eee
91. nd Optimization 45 Optimizing Signals from the Third Laser Intercept System configuration can vary greatly If your system has a triple laser beam splitter use this procedure to optimize the signal detected by detector option 3 DO3 or detector option 4 DO4 located to the left of OBS2 See Figure 2 5 on page 39 Preparing for Third Laser Optimization 1 Right click the dated specimen and choose New Tube name the new tube UV This tube will be used to optimize signals from the GU Feb 2006 third laser intercept Your experiment should look 0 wnat Settings ask 8 Global Worksheets similar to that shown in the figure at the right 3 022306 488 nm In the Parameters tab deselect the Log checkbox for mE fluorescent signals detected from the UV laser Create a new global worksheet On the new worksheet create appropriate plots for the UV tube For example create the following plots e FSC A vs UV1 A and FSC A vs UV2 A dot plots e UV1 A and UV2 A histograms Right click on any plot and choose Create Statistics View Edit the statistics view to display the mean and CV for each UV channel On the Population tab deselect Events and Parent On the Statistics tab show the mean and CV for UV parameters In the Acquisition Dashboard set the Events to Record to 10 000 events and the Events to Display to 500 events 46 BD FACSVantage SE Digital Option User s Guide 8 Select the UV tube in the Browser in
92. ngs optimizing 54 seven color configuration 156 sheath pressures optimal frequency with 100 shortcuts keyboard 76 signals low area 145 no fluorescent 145 primary laser optimizing 32 34 second laser optimizing 39 41 third laser optimizing 46 47 troubleshooting 149 Single Cell mode 93 single stained controls 55 166 BD FACSVantage SE Digital Option User s Guide singlet population defining 122 optimizing CV 123 six color configuration 154 slides sorting into 109 software about 14 starting up 25 Sort Layouts about 81 creating 75 83 108 custom 75 113 editing 85 entering populations 83 examples 81 Sort Precision modes 4 Way Purity 92 about 88 creating 74 93 defaults 92 Fine Tune 93 Initial 93 Purity 92 Single Cell 93 Yield 93 sort rate troubleshooting 150 Sort Reports displaying 75 87 exporting 88 printing 88 103 Sort Setup controls about 75 Breakoff controls 78 button functions 77 Streams controls 79 sorting about 73 94 adjusting settings 76 99 beads for drop delay 106 collection devices 81 conflicts 84 86 88 controls 74 85 counters 86 into plates 109 into slides 109 into tubes 96 main adjustments 96 monitoring 87 109 pausing 85 109 populations 83 95 107 report 87 saving settings 80 103 setting up for 94 Sort button 85 starting 85 107 stopping 85 test mode 77 troubleshooting 150 starting acquisition 33 computers 25 instrument 24 sample flow 33 software 2
93. ngs Using Instrument Setup on page 54 Vv Tip When sorting perform sample optimization with the drop drive on 4 and the frequency at an appropriate level Use gating buttons and subsetting methods to define the population s of interest NOTICE Populations defined by snap to gates cannot be sorted Examples of gating analysis can be found in Recording and Analyzing Data on page 63 and in Getting Started with BD FACSDiva Software Chapter 4 Sorting 95 Main Sorting Adjustments Each of the following adjustments is explained in more detail in the context of each sorting example 1 Adjust the drop drive frequency for the shortest droplet breakoff 2 Adjust the amplitude to optimize the last connected drop 3 Adjust the phase to obtain single side streams 4 Use the Accudrop option to determine the drop delay Setting Up for Sorting Into Test Tubes This section describes how to set up for sorting into test tubes Use this procedure as a guide to set up similar sorting experiments For general guidelines see General Sorting Overview on page 94 Installing the Sorting Hardware AA Sorting hardware could be contaminated with biohazardous material Follow universal precautions when handling instrument hardware 1 Install an appropriately sized nozzle tip For guidelines see Setting Up for Sorting on page 94 NOTICE Instrument QC must be performed each time you change the nozzle tip The 100 um nozzle tip is not re
94. nstrument optics must match the current instrument configuration Modifications to the current configuration will not apply unless you click Set Configuration 2 Create a new experiment specimen and tube rename the experiment DNA M Tip To place the experiment inside an existing folder select the folder before creating the experiment 3 Rename the new specimen DNA OC Kit and rename the first tube CEN This tube will be used to optimize signals from the first amp Gl pna laser Your experiment should look similar to that shown Instr Settings DNA QC Kit in the figure at the right P cen 4 Click the Parameters tab in the Inspector and make the following changes e Delete all parameters except FSC SSC and PI e Select the height H and width W checkboxes for PI e Verify that the Log checkbox is deselected for all parameters Inspector x Instr Settings Parameters Threshold Ratio Compensation Parameter A Ww e FSC 250 Vv O O SSC 200 C Vv C C DAPI 500 Cr R T V 118 BD FACSVantage SE Digital Option User s Guide 5 Click the Threshold tab and change the threshold parameter to PI verify that the threshold value is set to 5 000 Inspector x Instr Settings Parameters Threshold Ratio Compensation And Parameter Yalue 6 Create the following plots for the CEN tube e FSC A vs SSC A dot plot e PI A vs PI H dot plot e PI
95. nterrogated drop will not be sorted Figure 4 7 Figure 4 7 Non target particle within a Purity Mask of 16 trailing drop drop being interrogated leading drop Nv Nv Purity Mask not Sorted Purity Mask non target particle If the Purity Mask were set to 8 for the same target particle the non target particle would fall outside of the Purity Mask so the interrogated drop would be sorted See Figure 4 8 NOTICE With any Purity Mask greater than zero the drop being interrogated must be free of contaminating particles or the drop will not be sorted Figure 4 8 Non target particle outside a Purity Mask of 8 trailing drop drop being interrogated leading drop Ly Purity Mask sorteo Purity Mask non target particle BD FACSVantage SE Digital Option User s Guide Phase Mask Particles near the drop edge can affect the breakoff and alter the trajectory of the deflected drop The Phase Mask restricts drop deflection when an event is too close to the edge of a drop or when there are events close to the edge of adjacent drops A Phase Mask is used to improve counting accuracy and side stream quality at the expense of yield For example when the Phase Mask is set to 16 the drop being interrogated will be sorted only if the target particle falls outside the Phase Mask Figure 4 9 Figure 4 9 Sorted and unsorted drop with Phase
96. nto two tubes BD FACSDiva software ignores the child events in both tubes Create a new subset under the parent population consisting of NOT Child Sort the child population into one tube and the NOT Child population into another tube Unable to move stage in Status tab Electronics mode set incorrectly Verify that the instrument is in digital mode Four way tube holder installed Remove the four way sorting hardware No stream when sorting onto a plate or slide Value for wrong stream entered in Streams tab Enter a value for the Far Left stream Insufficient stream deflection for four way sort Deflection angle adjusted incorrectly Increase the plate voltage e Change the angles of the tube holders e Lower the position of the four way tube holder e Place a spacer in the outer tube holders to raise the level of the outer tubes Cracked tubes with four way 15 mL tube holder Wrong tubes used Use only polypropylene tubes BD Catalog No 352096 with the four way tube holder Chapter 7 Troubleshooting 151 152 BD FACSVantage SE Digital Option User s Guide Appendix A Optical Configurations The diagrams in this appendix show how to set up the optical bench to match the default instrument configurations Use the following examples as a guide when setting up your own instrument configuration e Six Color Configuration on page 154 e Seven Color Configuration
97. o touch the deflection plates because this could 102 10 result in arcing sparking You might need to adjust the frequency amplitude phase or streams settings to optimize the angle of the streams Once optimized the plate voltage can be increased Click the Streams tab and adjust the 2nd 3rd and 4th drop settings to tighten the center stream and fine tune the side streams Mi Tip Generally settings of 20 10 and 5 are good starting values for the 2nd 3rd and 4th drops respectively If the 2nd drop must be set to zero to obtain a narrow center stream the frequency setting probably needs adjustment BD FACSVantage SE Digital Option User s Guide 11 12 13 14 15 Adjust the Plate Voltage knob and Stream deflection percentages to direct the streams into the tubes Press F12 to turn off Test Sort Calculate the drop delay See Calculating the Drop Delay on page 104 Optional Save the values in the Breakoff and Streams tabs Choose Instrument gt Sort Setup gt Save and enter an appropriate name such as 11 psi 2 way sort in the dialog box that appears Click OK to save the settings The settings can be recalled for use in a similar sorting experiment For more information see Saving and Recalling Sort Setup Values on page 80 Print a Sort Report e Choose Sort gt Sort Report e In the Sort Report Window choose File gt Print Report Sort Report File Page Setup Sort R
98. of the center and side streams When illuminated evenly the streams appear to sparkle Figure 4 15 on page 105 104 BD FACSVantage SE Digital Option User s Guide Figure 4 15 Illuminating the center and side streams Turn off Test Sort Turn off the stream lamps to better view the streams Install a sample tube filled with a dilute suspension of Accudrop beads 1 drop of beads in 0 5 mL sheath fluid click the current tube pointer to start acquisition Click the Parameters tab in the Inspector and adjust the FSC voltage to place the bead population at channel 125 000 Draw an interval gate that encloses the entire histogram Figure 4 16 Set the endpoints of the interval at 0 and 262 x 103 For an accurate setting ensure that the interval gate encompasses the entire bead population including doublets Figure 4 16 Accudrop bead population Chapter 4 Sorting 105 Sorting Beads to Determine the Drop Delay 1 Right click the Accudrop Beads tube choose New Sort Layout and set up the Sort Layout as follows a AccuDrop Beads Sort Layout_001 x Device Precision Target Events Save Conflicts k Tube X inti lt lli z a Lett Right AccuDrop B Continuous 2 Adjust the micrometer dial to obtain the brightest bead spot on the center stream 3 Move the emission filter in front of the camera 4 Adjust the Sample Differential knob to achieve a bead event rate of approximately 4 000 events second 5
99. on page 156 e Eight Color Configuration on page 157 This appendix also includes a blank configuration worksheet that can be photocopied and filled in for any custom configurations See Configuration Worksheet on page 159 153 Six Color Configuration Configuration D Current Configuration 6 color Configurations Parameters Configuration Modified Date Parameter Laser Detector 4 13 06 9 44 00 AM ssc _ h ssc 4306 9 44 00 AM FITC 4 FLA PE u FL2 PerCP Cy5 5 1 FL3 uvi 3 FL4 Uv2 3 FLS APC 2 FLE Add Set Duplicate FITC FL1 575 26 23032 7 PE FL2 Sa 560 SP 712 21 610 P JO M PerCP Cy5 5 FL3 405 20 UV1 FLA D HO APC FL6 j C 505 SP 660 20 485 22 UV2 FL5 s O 154 BD FACSVantage SE Digital Option User s Guide Alternate Six Color Configuration Five Colors DNA Configuration amp l Current Configuration 6 color alternate Configurations Parameters Configuration Modified Date Parameter Laser Detector 6 color 4 3 06 9 44 00 AM FSC 1 Fse 7 color 4 1306 9 44 00 AM ssc _ 1 ssc _ 8 color 4 13 06 9 44 00 AM FITC 1 FL1 B color alternate 1413 06 3 09 45 PM PE
100. oncentration Flow cytometry can be used to measure the concentration of intracellular free calcium ions Measurement of calcium ion Ca concentration can be made on large numbers of single cells which provides information about the number of responding cells as well as the relative magnitude of the response to a given stimulus Ca concentration can be correlated with other parameters such as time phenotype and cell cycle In their resting state eukaryotic cells maintain an internal Ca concentration far less than that of the extracellular environment Elevation in intracellular Ca concentration is often used as an indicator of cellular activation in response to a stimulus Calcium flux is also an indicator of whether the cells in a population remain functional after exposure to a drug or other compound Several fluorescent dyes measure intracellular Ca levels For most of them the amount of Cat entering a cell is indicated by a change in fluorescence emission For example the emission spectrum of indo 1 changes from blue to violet upon binding to Ca The ratio of violet to blue fluorescence is independent of the amount of dye within the cell When normal cells are analyzed for calcium flux with indo 1 by flow cytometry a shift in the violet blue ratio is obtained Figure 6 1 A break in data occurs when the stimulus is added to the sample tube The increase in the ratio over time reflects the increase in intracellular Ca
101. ontrols creating 57 components AccuDrop monitor 17 digital electronics module 16 digital oscilloscope 16 four way sorting 19 computer starting up 25 workstation 20 configurations alternate eight color 158 eight color 157 five colors DNA 155 instrument defaults 153 seven color 156 six color 154 variants 39 worksheet 159 conflicts sort about 88 counting 86 printing 87 saving 84 troubleshooting 150 control switch about 17 analog operation 19 digital operation 17 controls Breakoff controls 78 compensation 57 inactive 18 instrument 18 32 74 single stained 55 sort setup 75 sorting 74 85 Streams controls 79 conventions manual x counters sort 86 150 162 BD FACSVantage SE Digital Option User s Guide creating Analysis objects 67 compensation controls 57 custom devices 75 113 global worksheets 64 Sort Layouts 75 83 108 Sort Precision modes 74 93 custom devices creating 75 113 deleting 114 customer support xi cytometer See instrument D data analyzing 63 67 digital vs analog 21 gating 67 recording 63 66 delay drop 104 laser 42 48 deleting custom devices 114 sort populations 85 sort setup values 80 digital control switch 17 data vsanalog 21 oscilloscope 16 starting instrument in 24 digital option about 14 active controls 74 components 15 inactive controls 18 operation 17 workstation 20 DNA experiments about 116 five colors configuration 155 QC Part
102. optical stage might be configured differently from the examples shown in the figure Chapter 2 Instrument Setup and Optimization 39 Preparing for Second Laser Optimization 1 Adda new tube to the experiment name the tube GE Feb 2006 633 nm Lis Instr Settings Global Worksheets S 022306 This tube will be used to optimize signals from the 486 nen second laser intercept Your experiment should look ci similar to that shown in the figure at the right 2 Inthe Parameters tab deselect the Log checkboxes for the fluorescent signals detected from the 633 nm laser 3 Click the New Global Worksheet button to create a new global worksheet 4 On the new worksheet create appropriate plots for the 633 nm tube For example create the following plots e FSC vs APC and FSC vs APC Cy7 dot plots e APC and APC Cy7 histograms 5 Right click any plot and choose Create Statistics View 6 Edit the statistics view to display the mean and CV for the appropriate fluorescence channels On the Population tab deselect Events and Parent e On the Statistics tab show the mean and CV for APC and APC Cy7 7 Inthe Acquisition Dashboard set the Events to Display to 500 events Make sure the Events to Record is set to the default 10 000 events 8 Select the 633 nm tube in the Browser in the Inspector choose Global Sheet2 from the Global Sheet menu The specified worksheet will appear when you move the current tube pointer to the
103. ows 1 Home Pos 0 0 Columns al Farthest Pos 0 0 Go to Home Set Home Go Farthest Set Farthest t t da gt 4 Delete Apply Close 3 Select the text in the Name field and enter a new name 4 Enter the number of sort location Rows and Columns A device can have up to 60 rows and 25 columns Chapter 4 Sorting 113 5 Use the Arrow keys to move to the Home location click Set Home See Adjusting the Home Location on page 111 There are no default values for custom devices so more initial adjustment with the Arrow keys is required 6 Use the same procedure to move to the Farthest location click Set Farthest The Farthest sort location is the well or spot on the lower right corner of the collection device 7 Click Apply and then click Close After you set the Home and Farthest locations custom devices are listed in the Device drop down menu in the Sort Layout window NOTICE Once custom devices are defined you cannot change the numbers of rows and columns 8 Proceed with Sorting on page 107 Deleting a Custom Device 1 Choose Sort gt Custom Devices 2 Select the name of the device to be deleted from the list of custom devices Figure 4 20 on page 113 3 Click Delete The device is deleted from the custom device list but is retained within any Sort Layouts where it was used 114 BD FACSVantage SE Digital Option User s Guide 5 DNA Analysis The following topics are cov
104. p delay 106 experiment 104 monitor 17 104 acquisition events to record 64 starting 33 troubleshooting 144 adjusting area scaling 35 43 49 125 Home location 75 111 laser delay 42 48 sort settings 76 99 streams 33 79 threshold 59 voltages 59 window extension 42 48 alignment sample preparing 26 Alpha control 32 amplitude about 78 optimizing 100 analog data vs digital 21 operation 19 oscilloscope 17 34 analysis calcium flux data 139 data 63 DNA data 127 immunophenotyping 67 primary laser results 37 reusing 70 second laser results 45 third laser results 51 applications calcium flux 130 DNA 116 sorting 94 area scaling adjusting 43 49 DNA experiment 125 primary laser 35 troubleshooting 145 149 assistance technical xi Attenuation 77 beads AccuDrop 104 alignment 26 sorting for drop delay 106 161 biohazardous hardware 94 waste 24 breakoff about 78 adjusting settings 99 adjusting with keyboard 76 100 controls 78 optimal distance 100 C calcium flux about 130 data analysis 139 experiment 132 measuring 137 optimization 131 optimizing sample 135 Time parameter 131 calculating compensation 62 drop delay 104 calf thymocyte nuclei CTN preparing 117 resolution 124 running 124 CellQuest Pro viewing 20 chicken erythrocyte nuclei CEN experiment 118 optimization 117 preparing 117 running 120 coefficient of variation CV high 148 compensation calculating 62 c
105. pulation using another gate type Unusual pattern on digital oscilloscope while setting phase Test Sort off Turn on Test Sort and then push the AUTOSET button on the oscilloscope control panel Sort button disabled Current tube pointer not set to current tube Click to set the current tube pointer next to the appropriate tube Sort Layout counters not updating Viewing Sort Layout for another tube Double click the appropriate tube in the Browser to view worksheet objects for that tube High sort conflict rate Event rate too high for drop drive frequency Decrease the event rate Sorting parent and child populations into two different tubes Verify the gating hierarchy Purity Mask too high Decrease the Purity Mask Unexpected sort rate Wrong stream displayed in Sorting status bar Use the arrow buttons to choose the appropriate stream See Using Sorting Controls on page 85 Laser noise Decrease the drop drive amplitude Erratic sort rate Event rate too high Decrease the event rate 150 BD FACSVantage SE Digital Option User s Guide Sorting Troubleshooting continued Observation Unexpected sort results Possible Causes Incorrect logic in population hierarchy Recommended Solutions Verify the gating strategy Sorting parent and child populations into two different tubes If you try to sort a parent and its child population i
106. r right and right streams by changing the amount of charge applied to sorted droplets from 0 100 The far right stream is used for four way sorting the right stream is used for two and four way sorting e 2nd 3rd 4th Drop apply a correction factor for the drop charge as a percentage of the previous drop from 100 to 100 NOTICE BD recommends that the amount of charge applied to the far left and far right streams not exceed 80 When the charge is set to 100 there is no remaining charge available for stream shaping via the 2nd 3rd and 4th drop correction factors Chapter 4 Sorting 79 Saving and Recalling Sort Setup Values Sort setup values settings in the Breakoff and Streams tabs are automatically saved when you quit the application When you restart the most recently used set of values is retained You can save sort setup values for different sorting applications using the Sort Setup option on the Instrument menu For example you might want to define standard settings for different sorting pressures or sets of values for a two way or four way sort See Adjusting Sort Settings on page 99 for an example e Choose Instrument gt Sort Setup gt Save As to save a set of sort setup values Choose High Medium Low or Custom or enter a new name and click OK NOTICE Choosing High Medium Low or Custom will overwrite the existing settings e Choose Instrument gt Sort Setup gt Recall to switch between predefined
107. re 1 5 Four way tube holder Chapter 1 Features of the Digital Option 19 20 BD FACSDiva Workstation The BD FACSDiva workstation controls the BD FACSVantage SE flow cytometer when operated in digital mode It consists of a Windows based computer running BD FACSDiva acquisition and analysis software Refer to the BD FACSDiva Software Reference Manual for complete instructions on using the software The BD FACSDiva workstation is operated independently of the BD FACStation workstation with a separate keyboard and mouse Viewing BD FACSDiva and BD CellQuest Pro Software Simultaneously During digital operation BD FACSDiva software can be expanded and viewed on both monitors simultaneously Alternatively as shown in Figure 1 6 signals can be viewed in BD FACSDiva software on one monitor and BD CellQuest Pro software on the other monitor by pressing the appropriate pushbutton on the bottom of the BD FACStation monitor Your service engineer will indicate the appropriate button during installation NOTICE The monitors included with your system might be different from those shown in the figure Figure 1 6 Viewing BD FACSDiva and BD CellQuest software simultaneously o 0090000 10 gt 0080000 10 BD FACSVantage SE Digital Option User s Guide If you are accustomed to working with analog signals note that digital data looks very different See Figu
108. re 1 7 Figure 1 7 Digital vs analog data Data 002 Bead data J PE Beads PE FL2 H i a gt 1 FITC Beads Negatives FITC FL1 H BD FACSDiva plot BD CellQuest Pro plot A Use caution when viewing digital data within BD CellQuest software Data viewed in BD CellQuest software results from instrument setting adjustments made in BD FACSDiva software Digital instrument settings are not updated in BD CellQuest instrument settings files or the resulting FCS files BD does not recommend saving BD CellQuest files during digital operation use BD CellQuest signals for troubleshooting purposes only Chapter 1 Features of the Digital Option 21 22 BD FACSVantage SE Digital Option User s Guide 2 Instrument Setup and Optimization This chapter describes how to start up the BD FACSVantage SE instrument for operation in digital mode and how to use BD FACSDiva software to optimize the instrument before acquisition The following topics are covered in this chapter e Starting Up the Instrument on page 24 Instrument Optimization and Quality Control on page 26 e Reusing the Experiment on page 52 23 Before beginning this chapter you should be familiar with the following e General instrument setup procedures refer to the BD FACS Vantage SE User s Guide for detailed information on instrument setup e BD FACSDiva workspace components e BD FACSDiva instrument and acquis
109. rifying Area Scaling for the Third Laser Because each laser has a different laser intercept height area scaling needs to be verified for each laser after its signal has been optimized Refer to the BD FACSDiva Software Reference Manual for more information about area scaling 1 Select the height H checkbox for the UV2 parameter 2 Create a histogram for UV2 H draw an interval marker around the fluorescent peak 3 In the Laser tab of the Instrument window adjust area scaling for the third laser until the UV2 A intensity is similar to the UV2 H intensity Figure 2 10 on page 50 4 Optional Delete the UV2 H histogram 5 Deselect the checkbox for the UV2 H parameter Chapter 2 Instrument Setup and Optimization 49 Figure 2 10 Third laser area scaling before left and after right adjustment Instrument x Instrument x Status Parameters Thveshold Compensation Ratio Laser Status Parameters Thveshold Compensation Ratio Laser 022306 UV 022306 UV 3 UV2 A A ET 3 35 w a a Oo 50 100 150 200 250 50 100 150 200 250 UV 2 A x 1 000 UV 2 A x 1 000 022306 UV 022306 UV N UV2 H e a o 50 100 150 200 250 50 100 150 200 250 UV 2 H 1 000 UV 2 H lt 1 000 50 BD FACSVantage SE Digital Option User s Guide Recording and Analyzing Third Laser Results 1 Adjust the voltages to display UV signal between 100 000 an
110. s In general the following settings are optimized for each sample type and fluorochrome used e FSC and SSC voltages e FSC threshold e FSC and fluorescence area scaling e fluorescence PMT voltages compensation Each adjustment is explained in detail in the sections that follow It is important that you adjust these settings in order as some adjustments influence others You might need to vary certain steps for different sample types BD FACS Vantage SE Digital Option User s Guide To demonstrate these adjustments the following sections describe how to optimize settings for a lysed washed whole blood sample LWB stained with the following mouse anti human antibodies In this example compensation will be automatically calculated using the Instrument Setup feature For more information about this feature refer to the BD FACSDiva Software Reference Manual If you are performing compensation manually not all steps will apply Unstained control FITC stained control PE stained control PerCP Cy5 5 stained control APC stained control APC Cy7 stained control Mouse IgG FITC Mouse IgG PE Mouse IgG PerCP Cy5 5 Mouse IgG APC Mouse IgG APC Cy7 CD8 FITC Mouse IgG PE Mouse IgG PerCP Cy5 5 Mouse IgG APC Mouse IgG APC Cy7 Mouse IgG FITC CD8 PE Mouse IgG PerCP Cy5 5 Mouse IgG APC Mouse IgG APC Cy7 Mouse IgG FITC Mouse IgG PE CD8 PerCP Cy5 5 Mouse IgG APC Mouse IgG APC Cy7 Mouse IgG FITC Mouse I
111. s properly adjusted when the UV signal intensity is at its highest M Tip Asa troubleshooting measure estimate the laser delay setting from the analog oscilloscope using the Special Setup feature in BD CellQuest software e Draw an interval gate around each peak on the UV histograms Use the population hierarchy to rename each population defined by the interval gates For example change P1 to UV1 and P2 to UV2 e Select the Laser tab in the Instrument window Adjust the third laser Delay setting in increments of 1 to obtain the highest mean channel for the UV populations Figure 2 9 Change the window extension to zero to capture pulses within the narrowest time window Adjust the third laser Delay setting in increments of 0 1 to obtain the highest mean channel for the UV populations Figure 2 9 Figure 2 9 Third laser delay before left and after right adjustment Instrument x Instrument Status mpen j Laser Status P z ren F Name Delay Area Scaling Name Area Scaling 1 0 00 oso h 0 80 2 15 90 0 70 2 3 2 1 00 ls 022306 UV 022306 UV w uv1 S 5w o o o oO Sea H 50 100 150 200 250 50 100 150 200 250 UV 1 A amp 1 000 UV 1 A 1 000 48 BD FACSVantage SE Digital Option User s Guide e Reset the window extension to the appropriate setting typically 2 A larger window extension allows more flexibility for capturing pulses Ve
112. s at its highest M Tip Asa troubleshooting measure estimate the setting from the analog oscilloscope using the Special Setup feature in BD CellQuest software Draw an interval gate around each peak on the fluorescence histograms Use the population hierarchy to rename each population defined by the interval gates For example change P1 to APC and P2 to APC Cy7 Select the Laser tab in the Instrument window Adjust the second laser Delay setting in increments of 1 to obtain the highest mean channel for the fluorescent populations Figure 2 6 on page 43 Change the window extension to zero to capture pulses within the narrowest time window Instrument xl Status Parameters Threshold Compensation Ratio Laser Name Delay Area Scaling 1 0 00 0 80 2 15 00 1 00 3 25 00 1 00 Window Extension 0 000 t FSC Area Scaling 1 00 BD Defaults Adjust the second laser Delay setting in increments of 0 1 to obtain the highest mean channel for the fluorescent populations Figure 2 6 on page 43 42 BD FACSVantage SE Digital Option User s Guide Figure 2 6 Second laser delay before left and after right adjustment EJ Instrumer xil TOMEI x Parameter Compensation Ratio Laser Status Parameters Th ion Ratio Laser Area Scaling 022306 633 nm APC Cy7 PC Cy7 Count TITI TIT TII teir 50 100 150 200 60 100 150 200 250
113. ser beam EXCITATION BEAM FOCUS 1 X control moves nozzle right and left 2 Y control moves nozzle away from and toward you 3 Theta lock prevents theta control from moving 4 Zcontrol moves nozzle up and down in an arc 5 Theta control moves nozzle along an arc so stream moves right and left 6 Alpha control moves nozzle along an arc so stream moves away from and toward you 7 Fluorescence channel height adjustment wheel raises and lowers fluorescence objective lens 8 FSC obscuration bar vertical adjustment moves FSC obscuration bar up and down 9 Excitation Beam Focus wheel moves beam focus lens to adjust laser beam focal point on the sample stream 10 Fluorescence Focus control knob moves objective lens to adjust focal point of the fluorescence image access through the upper side door BD FACSVantage SE Digital Option User s Guide Set or check the Z distance e Turn on the drop strobe e Use the camera vertical adjustment wheel to position the viewing mark at the laser intercept Figure 2 2 e If the laser intercept is not visible adjust the Y control e Adjust the Z control to position the nozzle at the upper reference mark Figure 2 2 Setting the Z distance nozzle upper reference mark viewing mark Check the trajectory of the fluid stream The stream should be entering the center or front third of the stream aspirator I
114. software user s guide for more information about area scaling 1 uu FF W N With the current tube pointer next to the 488 nm tube in the Browser click the Parameters tab in the Instrument window Select the height H checkbox for the FITC parameter Change the axis on the PerCP Cy5 5 histogram to FITC H Click the Laser tab in the Instrument window Adjust area scaling for the first laser until the FITC A intensity is similar to the FITC H intensity See Figure 2 3 on page 36 for an example Chapter 2 Instrument Setup and Optimization 35 36 Figure 2 3 Primary laser area scaling before left and after right adjustment Status Parameters Threshold Compensation Ratio Laser Area Scaling Status Parameters Threshold Compensation Ratio Laser Area Scaling 0 00 3 25 00 1 00 25 00 Window Extension 2 00 t FSC Area Scaling 1 00 BD Defaults 3 Window Extension 2 00 Sit FSC Area Scaling 10 sit 022306 488 nm 022306 488 nm 200 50 1 000 100 150 FITC A 022306 483 nm 200 250 1 000 6 Change the FITC H histogram plot to PerCP Cy5 5 A 7 Deselect the checkbox for the FITC H parameter BD FACSVantage SE Digital Option User s Guide 200 BD Defaults 250 1 000 Recording and Analyzing Primary Laser Results 1 Optional Adjust the voltages to display fluorescence signal between 100 000 an
115. t appear after selecting the sort location field s right click the selected fields to see the menu e Enter the number of target events and the population s for the remaining sort location fields if necessary 108 BD FACSVantage SE Digital Option User s Guide Starting and Monitoring the Sort 1 A Open the camera door and install the collection tubes plate or slide containing nutrient medium Install the sample tube on the cytometer and close the camera door Turn the Fluidics Control knob to Run Verify that the current tube pointer is indicating the appropriate tube in the Browser click Sort Optional Click Record Data to save data for the tube Sorting continues until the required number of cells has been sorted If the number of target events is set to Continuous sorting continues until you manually stop sorting Monitor the sort progress from the Sort Layout window The number of events sorted into each sort location appears in the corresponding field The Sort Rate Conflict Rate and number of conflicts are displayed in the counter fields See Using Counters on page 86 NOTICE To pause during sorting click the Pause button Sort counts are retained when you restart sorting by clicking the Pause button again After completing a four way sort remove the four way sorting hardware To avoid instrument damage do not start up the instrument with the four way sorting hardware installed Setting Up for
116. t the phase using the digital oscilloscope Aue ae gt Lau gt Pe om w e RSA na ER AUTOSET digital T mm Sel oo NY E i i Ez u T veu Sri be drop charge J W a voursov vocrsow Na y CHI 100V CH2 100V M250s CH1 7 SIN gt p g SEC DIV knob Chapter 4 Sorting 101 e Turn on Test Sort by clicking the Test Sort button e Press the AUTOSET button on the oscilloscope e Adjust the SEC DIV knob until you can clearly see the digital amplitude and drop charge waveforms e Adjust the Phase value in the Breakoff tab until the drop charge is synchronized with the top or bottom of the amplitude waveform Turn the Plate Voltage knob on the instrument control panel counter clockwise to its minimum setting Turn on the deflection plates using the push button on the instrument control panel A A To prevent shock do not touch the deflection plates when the red warning light appears on the control panel The plates remain energized even when the camera door is open Slowly turn up the plate voltage until the side streams are visible and deflecting away from the waste aspirator ANA Do not allow the streams t
117. tatistics view and click the Edit Statistics View button in the Inspector e Add the required statistics delete Events and Parent from the Population tab 9 Inthe Acquisition Dashboard set the Events to Record to 1 000 000 evt the Events to Display to 500 evt and the Stopping Time to 360 sec EB Acquisition Dashboard x c Current Activity Active Tube vVell Threshold Rate Stopping Gate Events Elapsed Time Cal 0 evtis 0 evt 00 00 00 Basic Controls gt Next Tube amp Acquire Data 5 Record Data Z Acquisition Setup Storage Gate M ateverts Events To Record 1000000 evt v Stopping Time sec gt 360 YH Stopping Gate MN al Everts 7 Events To Display 500 evt NOTICE Only the specified number of events is displayed in plots during acquisition and recording After data recording is complete all recorded events will be displayed 134 BD FACSVantage SE Digital Option User s Guide Optimizing the Calcium Sample 1 Install the unstimulated sample on the cytometer turn the Fluidics Control knob to Run Verify that the current tube pointer is in front of the Ca 1 tube in the Browser click once on the pointer to start acquisition Events appear in the plots Adjust the sample differential to obtain an event rate of approximately 1 000 events second The event rate is displayed in the Acquisition Dashboard While viewing the FSC vs SSC plot Figure 6 3 make t
118. te name For example use 5 Color Expt or your initials followed by an appropriate identifier 56 BD FACSVantage SE Digital Option User s Guide 5 Select the experiment level instrument settings in the Browser click on the Parameters tab and delete any unnecessary parameters Figure 3 1 For this example delete all parameters except FSC SSC FITC PE PerCP Cy5 5 APC and APC Cy7 To delete parameters press the Ctrl key and click the selection button next to each parameter that is not used in the experiment When all rows have been selected press the Delete key or click the Delete button in the Inspector To change to a different parameter than what is listed click the parameter name and choose a different fluorophore from the drop down menu Figure 3 1 Parameters for five color optimization Instrument xi Status Parameters Threshold Compensation Ratio Laser Parameter PE PerCP Cy5 5 APC JV 7 77 7771 TVITI I79 779 77 77 7771 qalalla APC Cy7 6 Choose Instrument gt Instrument Setup gt Create Compensation Controls The Create Compensation Controls dialog box appears listing only those parameters specified in the previous step Figure 3 2 on page 58 7 Click OK to add the specified controls Alternatively add and define label specific controls and then click OK Chapter 3 Running Samples 57 Figure 3 2 Creating compensation controls F
119. the Inspector choose Global Sheet3 from the Global Sheet menu The specified worksheet will appear when you move the current tube pointer to the UV tube Optimizing Signals from the Third Laser 1 Block the second beam intercept by closing the laser shutter Refer to the laser manufacturer s instructions Click to move the current tube pointer in front of the UV tube in the Browser and click Acquire Data Mi Tip If you do not see any signal increase the appropriate PMT voltage or change to Log If you still do not see any signal verify the Delay setting for the third laser see step 6 on page 48 NOTICE During digital operation use the analog pulses displayed on the analog oscilloscope for troubleshooting purposes only Adjust the appropriate beam splitter s to direct the UV signal to the appropriate PMT s Adjust the two rear rotators rear knobs to obtain the highest signal intensity and lowest CV on the UV plots The knobs are located on the beam steering prism assembly of the laser being optimized If necessary adjust the two front translators front knobs to obtain the highest signal intensity and lowest CV Minor adjustment of the translators might be needed after performing laser alignment The translators do not need adjusting on a daily basis Chapter 2 Instrument Setup and Optimization 47 6 Optimize the third laser delay Adjust the laser delay to synchronize laser signals in time The delay i
120. timizing the FITC signal Continue adjusting the controls and closing the iris until the iris is completely closed 9 With the iris closed adjust the Y control and Excitation Beam Focus wheel for maximum FITC signal 10 Compare the FITC signal intensity with the iris open and closed You should not lose more than half the maximum FITC signal intensity with the iris completely closed 11 Open the FL1 iris and adjust the beam splitters for maximum fluorescence intensity On the appropriate plots maximize the signal for SSC PerCP Cy5 5 and PE BD FACSVantage SE Digital Option User s Guide 12 13 Adjust the obscuration bars for minimum FSC and SSC noise if necessary Verify the trajectory of the fluid stream The stream should remain in the center or front third of the stream aspirator If necessary adjust the Alpha and Theta controls to correctly position the fluid stream After adjusting the controls repeat steps 6 through 12 Verifying Area Scaling for the Primary Laser BD FACSDiva software uses area as its default parameter The area measurement provides a complete measurement of the voltage pulse but it can be affected by how well the laser is focused and by the sheath pressure To ensure that the PMT works within its linear dynamic range it is important to adjust the height and area measurements to the same magnitude For accurate linearity verify area scaling each time you optimize laser signal Refer to the
121. ting Controls Sorting controls appear at the bottom of the Sort Layout window Use these controls to start pause resume and stop sorting events H Sort Pause e Sort starts sorting events for the current acquisition tube All counters reset to zero when this button is clicked Events are sorted until the requested number of sorted events has been reached Click the Sort button again to stop sorting before reaching the requested number of events the counters stop at the number of sorted events If you click Sort to restart sorting the counters reset to zero e Pause stops sorting but not acquisition sort counters freeze when the Pause button is clicked Click the Pause button again to continue sorting and to continue incrementing the sort counters Chapter 4 Sorting 85 Using Counters Counters provide ongoing status during sorting the fields cannot be edited To display fewer counters in the Sort Layout window click the View Counters button and choose a menu option The corresponding counter is hidden Only counters with a checkmark next to the name are displayed z Sort Sample Sort Layout x Device Precision Target Events Save Conflicts 2 Tube v Purity v v E Left Right Sort Rete Rate NA NA Confl Cnt NA NA Confl Rate NA NA NA NA H sort H Pause View Counters v Sort Rate v Conflicts Count v Conflicts Rate v Efficiency NOTICE Counters can
122. ting buttons and subsetting methods to define the population s of interest NOTICE Populations defined by snap to gates and those derived from them cannot be sorted Examples of gating analysis can be found in Recording and Analyzing Data on page 63 and in Getting Started with BD FACSDiva Software Chapter 4 Sorting 107 Setting Up the Experiment 1 In the Browser right click the tube containing the defined population subset s to be sorted and choose New Sort Layout Alternatively select a tube in the Browser and click the New Sort Layout button E in the Workspace toolbar By default the 2 Tube Sort Layout appears 2 Make appropriate entries in the Sort Layout az Sort Sample Sort Layout xj Device Precision Target Events Save Conflicts 4 Tube x Purity r r Far Left Left Right Far Right Sort Rate NA Confl Crt NA Confl Rate NA NA View Counters e Choose the collection device from the Device menu e Choose the precision mode from the Precision menu For four way sorting use 4 Way Purity mode Enter the number of target events by choosing a value from the drop down menu or entering a number in the field e Select the sort location field s to be sorted into Select multiple fields by dragging the mouse select a row or column by clicking the row or column header e Add the required population s to each sort location field If the Add menu doesn
123. ues are displayed from 26 262 143 Thus the first log decade ranges from 26 262 Gridlines are used to delineate log decades on plots 10 Optimize the voltages to place the negative population for each fluorescent parameter within the first log decade Figure 3 5 on page 61 Refer to the BD FACS Vantage SE User s Guide if you need assistance optimizing the fluorescent signal 60 BD FACSVantage SE Digital Option User s Guide Figure 3 5 Unstained control tube after PMT adjustment Unstained Control 50 100 150 200 250 FSC A x 1 000 Count Unstained Control FITC A Unstained Control Unstained Control Count Count PE A 10 10 PerCP Cy55 A Unstained Control Count Unstained Control 102 10 10 10 APC Cy7 A 11 Click Record Data when all events have been recorded remove the unstained control tube from the cytometer A Do not change the PMT voltages after the first compensation control has been recorded In order to calculate compensation all controls must be recorded with the same PMT voltage settings If you need to adjust the PMT voltage for a subsequent compensation control you will need to record all compensation controls again Chapter 3 Running Samples 61 Calculating Compensation Before you can calculate compensation you need to record data for each single stained control 1 Install the first stained control tube
124. ult Global Worksheet is enabled in User Preferences default option the worksheet is already present Expand the Global Worksheets folder to locate and rename the worksheet e If the Default Global Worksheet preference is disabled create a worksheet by clicking the New Global Worksheet button in the Browser toolbar You can create up to 50 global worksheets per experiment 4 Use the Experiment Layout window to define labels and to specify the number of events to record for each tube Figure 3 7 on page 65 Parameter labels will appear on plot axes and in all statistics views e Choose Experiment gt Experiment Layout On the Labels tab enter appropriate labels for the tube For example enter CD4 in the FITC field use the Tab key to move to the next field 64 BD FACSVantage SE Digital Option User s Guide Figure 3 7 Entering parameter labels in Experiment Layout Experiment Layout Labels Keywords Acquistion Label Joos ie E 5 color Expt Lwe TBNK_001 PE PerCP Cyt PE PerCP Cyt TBHK_002 On the Acquisition tab enter 10 000 events for tubes 001 and 002 e Notice that the Acq tab in the Inspector updates automatically Experiment Layout Labels Keywords Acquisition Events to Record 10 000 E 5 color Expt Le _ TBHK_001 TBNK_002 e Click OK 5 On the global worksheet create appropri
125. ument configuration corresponds to the optical bench setup See Instrument Optimization and Quality Control on page 26 No sample in tube Add sample to tube or install new sample tube Sample not mixed properly Mix sample to suspend cells Sample tube cracked Replace the sample tube Threshold not set to correct parameter usually FSC Set the threshold to the correct parameter for your application 144 BD FACSVantage SE Digital Option User s Guide Acquisition Troubleshooting continued Observation No events in plots after clicking Acquire Data continued Possible Causes Multiple threshold parameters not set correctly Recommended Solutions Verify that the correct Boolean logic And Or was used for the threshold parameters Threshold channel too low or too high Adjust the threshold channel See Adjusting the Voltages and Threshold on page 59 Unexpected results after clicking Next Tube Current tube pointer on wrong tube Verify the current tube pointer is next to the tube you want to duplicate before you click Next Tube No fluorescent signal Current instrument configuration different from optical bench Verify that the current instrument configuration corresponds to the optical bench setup See Instrument Optimization and Quality Control on page 26 Wrong filter installed Make sure the appropriate filter is installed for each fluorochrome S
126. with BD FACSDiva Software For detailed information on software features refer to the BD FACSDiva Software Reference Manual BD FACSVantage SE cytometers modified with the digital option can be operated in analog mode as described in the BD FACS Vantage SE User s Guide Even when using the instrument in the digital mode consult the instrument user s guide for descriptions of instrument components daily shutdown maintenance and troubleshooting theory of operation and laser service procedures The BD FACS Vantage SE Digital Option User s Guide assumes you have a working knowledge of basic Microsoft Windows operation If you are not familiar with the Windows operating system refer to the documentation provided with your computer First time users of the digital option should read Chapter 1 to learn about option components Instructions for routine acquisition and analysis can be found in Chapters 2 and 3 For application specific information review Chapter 4 Sorting Chapter 5 DNA Analysis and Chapter 6 Calcium Flux For a summary of optical bench layouts for the default instrument configurations see Appendix A Conventions The following tables list conventions used throughout this guide Table 1 lists the symbols that are used to alert you to a potential hazard Text and keyboard conventions are shown in Table 2 Table 1 Hazard symbols Symbol Meaning Caution hazard or unsafe practice that could result
127. xperiment xi xi 13 14 15 16 19 20 23 24 26 26 27 32 39 46 52 Chapter 3 Running Samples 53 Optimizing Settings Using Instrument Setup 54 Creating the Experiment 0 0 0 cece cc ecc eco rece cee 56 Adjusting the Voltages and Threshold Qc 59 Calculating Compensation 0 cece eee eee eens 62 Recording and Analyzing Data 0 cece cece eee eee ee eees 63 Setting Up the Global Worksheet Quc 64 Recording Datars se 4 00280 eae bw 2A VR dp Heide ia 66 Analyzing Datare seien Biel d Oe GR RON ee BRA a ieee 67 Saving the Analysisos 5020542692 obit Sees Seats Sod Sean oh 70 Chapter 4 Sorting 73 Sorting Controls erer Witte rai hd e said re tates etal 74 Sort Setup Controls oii cious 3422404777 74 2848 26 4 4 4470 ic 75 Sort Layout ss tla ee seesaw Met Can das Eons 81 SREDO yo 2 2 9 0 EA D eee Meee EA eRe ee eet aes UR eee ye La 87 Conflict Resolution with BD FACSDiva Software 88 Yield Mask 2 54 isaaecd s dbdbaw eiae eaa E da edness 89 Purity Mask oss etnies os oie Sa tes 3S Cans Ge BAN ek 9G wen 90 Phase Mask acc rated Ae EE a ea ates Bales ee Lae 91 Sort Precision Modes s a c s 0 cece cece ence eee ee nee ences 92 General Sorting Overview roni a 52 ebau a 9 0 B N 30 9 66 B d 08 4 E A 94 Setting UpforSorting o coec ee eee eee 94 Main Sorting Adjustments cece ee
128. y laser signal 32 34 second laser signal 39 41 third laser signal 46 47 oscilloscope analog 17 34 digital 16 troubleshooting 150 P parameters labels 64 scatter distorted 148 Time 131 pausing sorting 85 109 phase about 78 optimizing 101 Phase Masks about 91 Yield Masks using with 90 91 plates sorting into 109 Index 165 plots excessive debris 148 noeventsin 144 unexpected events in 146 populations AccuDrop bead defining 104 sorting 83 95 107 troubleshooting 149 150 Precision Modes 88 92 See also Sort Precision modes preferences user 70 primary laser optimizing signals 32 34 results 37 verifying area scaling 35 printing Sort Reports 88 103 Purity Masks 90 Purity mode 92 Q quality control 26 R recording calcium flux data 137 compensation Tubes 61 62 data 63 66 DNA data 127 primary laser results 37 second laser results 45 third laser results 51 reports sort 87 results troubleshooting 145 151 reusing analyses 70 Right stream 79 S sample alignment 26 tubes four way sorting 97 98 sample optimization experiment 56 LWBexample 55 samples running 66 Save Conflicts 84 saving sort conflicts 84 sort setup values 80 103 scaling area DNA experiment 125 primary laser 35 second laser 43 third laser 49 troubleshooting 145 149 scatter parameters distorted 148 second laser adjusting delay 42 optimizing signals 39 41 results 45 verifying area scaling 43 setti
129. y polypropylene tubes BD Catalog No 352096 not polystyrene tubes BD recommends placing the 15 mL tubes into the two center tube holders only and using the outer tube holders for smaller tubes 1 Verify that the Fluidics Control knob is set to Off and that the deflection plates are not charged A A To prevent shock do not touch the deflection plates when the red warning light appears on the control panel 2 Install the adjustable support bracket below the center stream aspirator using the two metal thumbscrews 3 Install the pegs into the fifth and eighth holes from the top of the support bracket slide the four way tube holder onto the pegs Figure 4 12 NOTICE Optimize the position of the tube holder and the angle of the tubes for your system Figure 4 12 Installing the four way tube holder lo EXCITATION dea Focus support bracket tube holder installed 98 BD FACSVantage SE Digital Option User s Guide Adjusting Sort Settings 1 2 Click the buttons to turn on Drop Drive and Input or recall approximate breakoff settings for the sheath pressure Click the Breakoff tab in the Sort Setup window and input approximate settings Figure 4 13 If similar settings were saved recall them from the Instrument gt Sort Setup menu Figure 4 13 Preliminary breakoff settings for standard upper and high lower pressure Streams Frequency F1 27 0 Ytl Amplitude F2
130. yping analysis 67 experiment 64 Initial mode 93 Inspector troubleshooting 148 installing four way sorting hardware 97 nozzle tips 94 96 plate sorting hardware 110 two way sorting hardware 97 instrument controls 18 32 74 default configurations 153 disconnect error 142 not responding 143 optimization 26 starting up 24 intracellular calcium concentration 130 K keyboard shortcuts 76 troubleshooting 76 150 164 BD FACSVantage SE Digital Option User s Guide L labels parameter 64 label specific tubes 57 lasers delay 42 48 primary optimizing signals 32 34 QC results 37 45 51 second optimizing signals 39 41 third optimizing signals 46 47 Layout Sort See Sort Layouts Left stream 79 linearity DNA experiments 116 low area signal 145 M main sorting adjustments 96 Masks about 88 default Precision Modes 92 Phase 91 Purity 90 Yield 89 Master DAQ overflow error 142 measuring calcium flux 137 modes defining 93 Sort Precision 88 92 monitoring sorts 87 109 monitors AccuDrop 17 104 computer 20 N nozzle tips installing 94 96 optimal frequency with 100 sizes and catalog numbers 95 0 obscuration bar 32 on off switch about 17 analog operation 19 digital operation 17 optical configurations 153 optimization AccuDrop 104 CEN 117 CV of singlet population 123 for calcium samples 131 135 for DNA samples 116 127 for LWB samples 55 instrument QC 26 27 instrument settings 54 primar

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