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BD LSR II User's Guide

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1. Detector Array PMT mon gpass Bandpass Fluorochrome or Dichroic Laser Detector Filter Scatter Parameter Mirror violet octagon A 630 LP 655 8 Qdot 655 405 nm laser B 595 LP 605 12 Qdot 605 C 575 LP 585 15 Qdot 585 D 545 LP 560 20 Qdot 565 E 475 LP 525 50 AmCyan Qdot 525 F blank 450 50 Pacific Blue G blank blank none H blank none UV trigon A 505 LP 530 30 Indo 1 Blue 325 nm or i 355 nm laser B blank 450 50 Indo 1 Violet DAPI C blank none red octagon A 755 LP 780 60 APC Cy7 633 nm laser B 710 LP 730 45 Alexa Fluor 700 C 675 LP 685 35 Alexa Fluor 680 D 660 20 APC E blank blank none F blank blank none G blank blank none H blank none 188 BD LSR II User s Guide Optional Default Configuration Trigon and Octagon Maps If a slot contains a filter or mirror a number appears in the corresponding position on the configuration map If a slot contains a blank optical holder nothing is written in the corresponding position on the configuration map The default configuration maps are 6 Color Blue Octagon Default Configuration Map on page 190 2 Color Violet Trigon Default Configuration Map on page 191 6 Color Violet Octagon Default Configuration Map on page 192 2 Color UV Trigon Default Configuration Map on page 193 3 Color Red Trigon Default Configuration Map on page 194 4 Color Red Octagon Default Configuration Map on page 195 Appendix F Optional D
2. Adjust the DAPI voltage to place the singlet nuclei at approximately channel 50 x 103 on the DAPI A axis in the DAPI A histogram plot as in Figure 4 4 on page 90 Adjust the event rate displayed in the Acquisition Controls frame to approximately 200 events second with the SAMPLE FINE ADJ knob Draw Interval gates around the first two peaks on the DAPI A histogram Name the populations Singlets and Doublets Figure 4 4 on page 90 Chapter 4 DNA Analysis 89 Figure 4 4 Defining Singlet and Doublet populations 10739 164 Experiment Name Specimen Name Tube Name Collection Date DNA QC KitCEN Singlets Doublets fi 50 100 150 200 250 DAPI A DNA DNA QC Kit CEN Nov 21 2002 2 22 29 PM x 1 000 DAPI V DAPI YY Mean cy 84 162 23 6 67 466 1 1 78 758 12 9 DAPI A DAPLA Mean cy 145 799 57 5 48 538 96 170 Population DO All Events EX Singlets EX Doublets CV lt 3 8 In the Acquisition Controls frame click Record 9 When recording has completed note the CV of the Singlet population e If the CV is lt 3 continue to step 10 e Ifthe CV is gt 3 restart the acquisition Decrease the flow rate with the SAMPLE FINE ADJ knob until the CV is lt 3 and then re record the data If the CV does not improve see Troubleshooting on page 121 9 BD LSR II User s Guide 10 Verify the linearity and print the worksheet 11 e Divide the mean of the Doublet population by the me
3. Sample injection tube clogged Remove the sample tube to allow backflushing then run a tube of warm DI water for 20 minutes If the event rate is still erratic clean the sample injection tube See Daily Cleaning and Shutdown on page 108 Chapter 7 Troubleshooting 127 Instrument Troubleshooting continued Observation Possible Causes Erratic event rate Sample tube cracked Recommended Solutions Replace the sample tube Bal seal worn Replace the Bal seal See page 118 Sample injection tube clogged Remove the sample tube to allow backflushing then run a tube of warm DI water for 20 minutes If the event rate is still erratic clean the sample injection tube See Daily Cleaning and Shutdown on page 108 Contaminated sample Prepare the specimen again Ensure that the tube is clean Worn sheath filter Replace the filter See Changing the Sheath Filter on page 116 128 BD LSR II User s Guide Instrument Troubleshooting continued Observation Distorted scatter parameters Possible Causes Instrument settings maladjusted Recommended Solutions Optimize the scatter parameters Refer to the BD FACSDiva Software Reference Manual for instructions Air bubble in sheath filter or flow cell Purge the air from the filter See Removing Air Bubbles on page 49 Flow cell dirty Perform the system flush procedure See page 111 Air leak at sheath cont
4. Software issue Refer to the BD FACSDiva Software Reference Manual for more information on setting gates Air in sheath filter Purge the filter See Removing Air Bubbles on page 49 No sample in tube Add sample to tube or install new sample tube Sample not mixed properly Mix sample to suspend cells Waste tank full Empty the waste tank PMT voltages set too low or too high for display parameter Reset PMT voltages Too few events displayed Increase the number of events to display Chapter 7 Troubleshooting 123 Instrument Troubleshooting continued Observation Possible Causes No events in acquisition Sample injection tube clogged display and green RUN button continued Recommended Solutions Remove the sample tube to allow backflushing then run a tube of warm DI water for 20 minutes If the event rate is still erratic clean the sample injection tube See Daily Cleaning and Shutdown on page 108 Bal seal worn Replace the Bal seal See page 118 Laser not warmed up Wait the recommended amount of time for the laser to warm up e 30 min for the 488 nm blue e 60 min for the 325 nm UV e 30 min for the 355 nm UV e 15 min for the 405 nm violet e 20 min for the 633 nm red Laser delay set incorrectly Adjust the laser delay settings See Setting Laser Delay on page 203 Laser not functioning Verify malfunction by changi
5. A printed copy of the following document is distributed with the BD High Throughput Sampler HTS option BD High Throughput Sampler User s Guide The BD High Throughput Sampler User s Guide describes how to set up and operate the BD High Throughput Sampler HTS option It also contains a description of BD FACSDiva software features specific to the HTS BD FACSDiva Software Help Menu The following manual is available as a PDF from the BD FACSDiva Software Help menu BD FACSDiva Software Reference Manual The BD FACSDiva Software Reference Manual describes how to use BD FACSDiva software It includes instructions or descriptions for installation and setup workspace components acquisition controls analysis tools and data management BD LSR II User s Guide BD Biosciences Website Download The following white paper can be downloaded from the BD Biosciences website BD FACSDiva Option White Paper The BD FACSDiva Option White Paper contains an in depth discussion of the digital electronics used in the BD LSR II cytometer Instructions for downloading this document can be found at BD FACSDiva Option White Paper on page 146 About This Guide XV Technical Assistance For technical questions or assistance in solving a problem e Read sections of the documentation specific to the operation you are performing see BD LSR II Documentation on page xiii e See Chapter 7 Troubleshooting If additional assistanc
6. violet trigon 505 LP 525 50 AmCyan 405 nm laser blank 450 50 Pacific Blue QO we gt oO aol al mm UlO ul gt blank none Appendix F Optional Default Configurations 177 Table F 2 6 Blue 2 Violet 0 UV 3 Red default mirror and filter configuration continued Detector Array PMT Ba gpass Bandpass Fluorochrome or Dichroic Laser Detector Filter Scatter Parameter Mirror red trigon A 755 LP 780 60 APC Cy7 633 nm laser B 710 LP 730 45 Alexa Fluor 700 C 660 20 APC 6 Blue 0 Violet 2 UV 3 Red Configuration 6 Blue 0 Violet 2 UV 3 Red specifies a blue octagon and UV and red trigons Table F 3 on page 179 shows the detectors filters and mirrors used in the default configuration The word blank indicates that a blank optical holder should be used instead of a mirror or filter A dash indicates that no slot exists for a mirror in that PMT position The 6 Blue 0 Violet 2 UV 3 Red maps are e 6 Color Blue Octagon Default Configuration Map on page 190 e 2 Color UV Trigon Default Configuration Map on page 193 e 3 Color Red Trigon Default Configuration Map on page 194 178 BD LSR II User s Guide Table F 3 6 Blue 0 Violet 2 UV 3 Red default mirror and filter configuration Longpass Detector Array PMT Dichroic Bandpass Fluorochrome or Laser Detector z Filter Scatter Parameter Mirror blue octagon A 755 LP 780 60 PE Cy7 488
7. 10 Create a Statistics view and display the mean and CV for DAPI A and DAPI W e Select Populations gt Create Statistics View e Right click on the Statistics view and select Edit Statistics View Under the Population tab deselect Events and Parent Chapter 4 DNA Analysis 87 Figure 4 2 Setting up the Statistics view Ey Edit Statistics View E a Header Populations Statistics 57 efa ajajqjn eanna Decimal Places Sort by Parameter C Sort by Formula e Under the Statistics tab select the mean and CV for both DAPI A and DAPI W Figure 4 2 Set Decimal Places to 1 for the CVs e Click OK 11 Inthe Acquisition Controls frame set the Number to Record to 10 000 evt and the Events to Display to 500 evt HE Acquisition Controls Ea CEN Bevis 00 03 25 Next i Record Storage Gate O All Events v Stopping Gate 0 All Events v Events To Record 100e Events To Display som 88 BD LSR II User s Guide Running CEN 1 2 3 On the BD LSR II cytometer control panel press RUN and LO Install the CEN sample tube on the SIP Verify that the green Acquisition pointer is in front of the CEN Tube in the Browser In the Acquisition Controls frame click Acquire Adjust the FSC and SSC voltages to place the CEN on scale in the FSC vs SSC dot plot Figure 4 3 FSC and SSC voltages adjusted DNA QC KiLCEN 50 100 150 200 250 FSC A 1 000
8. O MV E In a four decade log display values are displayed from 26 262 143 The first log decade ranges from 26 262 Chapter 3 Running Samples 65 8 For each fluorescence parameter adjust the PMT voltages to place the negative population within the first log decade In the Parameters tab in the Instrument frame use the controls to adjust voltage settings Figure 3 11 Figure 3 11 Adjusting fluorescence PMT voltage Status Parameters Threshold Compensation Ratio Laser _Votage Log a H w Instrument Connected When all parameters are adjusted your fluorescence parameter data should look similar to that in the histograms of Figure 3 12 on page 67 66 BD LSR II User s Guide Figure 3 12 Unstained Control Tube worksheet plots after PMT adjustment SSC A 1 000 Unstained Control 50 100 150 200 FSC A 250 x 1 000 Unstained Control Unstained Control 150 190 Unstained Control Ss gt o o o o amp 10 10 104 105 10 10 10 10 PEA PerCP A Unstained Control 3 4 i gt o o 10 10 10 10 APC A Chapter 3 Running Samples 67 68 Recording Compensation Tubes Before you calculate compensation settings you need to record data for the unstained control and for each single stained control 1 Click the Record button in the Acquisition Controls frame to begin recording data for the Unstained Control
9. 1 Install the first sample tube on the cytometer 2 Move the Acquisition pointer to Beads_001 3 Click the Acquire button in the Acquisition Controls frame to begin acquisition While data is being acquired e Draw a gate around the singlets on the FCS vs SSC plot Use the Inspector to set the other plots to show only the singlet population Inspector x Pict Title Labets Dot Piot Tube yExperiment FourColorBeads Beads_001 X Parameter Frrc a v Y Parameter Percr a v Biexponential I x Axis Ty Axis I Show Grid Background Color Population Tr 4 Inthe Acquisition Controls frame click Record to record data 72 BD LSR II User s Guide 5 When event recording has completed remove the first tube from the cytometer The MyData worksheet plots should resemble those in Figure 3 14 on page 74 6 Install the second sample tube on the cytometer 7 Move the Acquisition pointer to Beads_002 8 Click the Acquire button in the Acquisition Controls frame to begin acquisition Before recording preview the data on the MyData worksheet 9 Inthe Acquisition Controls frame click Record to record data 10 When event recording has completed remove the second tube from the cytometer If you are recording more that two tubes repeat steps 6 through 10 for the additional tubes 11 Print the Experiment level Instrument Settings Right click the Instrument Settings icon and selec
10. As cells or other particles pass through a focused laser beam they scatter the laser light and can emit fluorescence Because the laser beam is focused on a small spot and particles move rapidly through the flow cell the scatter or fluorescence emission has a very brief duration only a few microseconds This brief flash of light is converted into an electrical signal by the detectors The electrical signal is called a pulse Figure A 10 1 A pulse begins when a particle enters the laser beam At this point both the beam intensity and signal intensity are low 2 The pulse reaches a maximum intensity or height when the particle reaches the middle of the beam where the beam and signal intensity are the brightest The peak intensity or height of the pulse is measured at this point 3 As the particle leaves the beam the pulse trails off below the threshold Figure A 10 Anatomy of a pulse __ signal intensity time signal intensity time signal intensity time 144 BD LSR II User s Guide Pulse Measurements The pulse processors measure pulses by three characteristics height area and width Figure A 11 Figure A 11 Pulse measurements height mmm a ee threshold baseline OV I A _ Yi time m I voltage E window gate E width e pulse height is the maximum digitized intensity measured for the pulse e pulse area is an integration of the digitized measures over time e pulse wi
11. experiment criteria 82 handles 27 spares kit filters 169 mirrors 169 symbols and labels xxi Instrument Configuration Application ICA 197 202 Instrument Configuration dialog 57 instrument configuration verifying 56 Instrument Setup feature 55 instrument handles 32 intracellular calcium concentration 96 J JDS Uniphase HeNe 33 K Kimmon HeCd 33 knob SAMPLE FINE ADJ 28 sheath clamp shown 46 L labels on instrument xxi laser classification xviii laser delay 203 optimizing 203 setting 203 lasers description 33 maintenance 112 power 33 quality control QC 51 160 safety xvii warming up 40 warmup times 33 Lightwave Xcyte 33 linearity DNA experiments 82 longpass LP filters 34 138 longpass dichroic mirror holder 42 longpass dichroic mirrors diameter 45 incident angle 45 minimum clear aperture 45 thickness 45 LSR II components 26 M maintenance fluidics daily cleaning 108 flushing system 111 periodic replacing Bal seal 118 replacing sample tube O ring 120 replacing sheath filter 116 117 scheduled battery change 114 battery test 113 lasers 112 system flush 111 waste management system 113 measuring calcium flux 104 Microsoft Windows operating system 37 mirrors dichroic longpass 34 extra 169 specifications 45 N NCCLS documents xx negative population placement 66 O octagon description 32 detector 26 location 44 shown 35 template 155 optical bench components 32 Index 211
12. C 655 LP 660 20 PE Cy5 D 600 LP 610 20 PE Texas Red E 550 LP 575 26 PE F 505 LP 530 30 FITC Alexa Fluor 488 G blank 488 10 SSC H blank none 180 BD LSR II User s Guide Table F 4 6 Blue 2 Violet 2 UV 3 Red default mirror and filter configuration continued Longpass Detector Array PMT Dichroic Bandpass Fluorochrome or Laser Detector Filter Scatter Parameter Mirror violet trigon A 505 LP 525 50 AmCyan 405 nm laser B blank 450 50 Pacific Blue C blank none UV trigon A 505 LP 530 30 Indo 1 Blue 325 nm or 355 nm laser B blank 450 50 Indo 1 Violet DAPI C blank none red trigon A 755 LP 780 60 APC Cy7 633 nm laser B 710 LP 730 45 Alexa Fluor 700 C 660 20 APC 6 Blue 6 Violet 0 UV 3 Red Configuration 6 Blue 6 Violet 0 UV 3 Red specifies blue violet octagons and a red trigon Table F 5 on page 182 shows the detectors filters and mirrors used in the default configuration The word blank indicates that a blank optical holder should be used instead of a mirror or filter A dash indicates that no slot exists for a mirror in that PMT position The 6 Blue 6 Violet 0 UV 3 Red maps are e 6 Color Blue Octagon Default Configuration Map on page 190 e 6 Color Violet Octagon Default Configuration Map on page 192 e 3 Color Red Trigon Default Configuration Map on page 194 Appendix F Optional Default Configurations 181 Table F 5 6 Blue 6 Violet
13. SIP 134 cleaning 108 components 29 30 problems with 122 replacing Bal seal 118 replacing sample tube O ring 120 sample injection tube 30 sample optimization 55 about 55 Experiment 59 single stained controls 56 70 sample tube requirements 122 samples running 72 saving analyses 79 scatter light 135 parameter distortion 129 setting laser delay 203 sheath clamp knob 46 container 31 46 container vent valve 110 filter replacing 116 117 flow 134 fluid 134 backflush 30 sheath pressure 134 shortpass SP filters 34 139 Show Grid checkbox 65 Show Population Hierarchy 75 shutdown instrument 108 side door 26 side scatter SSC 33 135 signals amplifying 35 Single Stained Setup dialog 69 singlet population discrimination 89 SIP See sample injection port Snap To polygon gate 64 software adjusting detector voltages instrument control spares filters and mirrors 169 kit 169 parts ordering 160 specifications filter and mirror 45 Specimen Calcium Flux setup 99 CEN optimization 86 spillover 141 stained control 62 statistics views 75 steering optics 32 35 Stokes shift 136 switch Battery Test 113 symbols on instrument xxi T technical assistance xvi temperature requirements xiv testing battery 113 text conventions xii thiazole orange TO 108 threshold adjusting 63 defined 147 setting 55 Threshold tab 63 Time parameter calcium flux 97 Index 213 trigon description 32 detector
14. by redness a tearing discharge from the mucous membrane lining the inner surface of the eyelid conjunctiva shedding of the corneal cell layer surface exfoliation and stromal haze These symptoms are associated with photokeratitis otherwise known as snow blindness or welder s flash which results from radiant energy induced damage to the outer epidermal cell layer of the cornea These effects can be the result of laser exposure lasting only a fraction of a second xvii Laser Classification Laser hazard levels depend on laser energy content and the wavelengths used Therefore it is impossible to apply common safety measures to all lasers A numbered system is used to categorize lasers according to different hazard levels The higher the classification number the greater the potential hazard The BD LSR II flow cytometer is a Class I 1 laser product per 21 CFR Subchapter J and Class 1 laser product per EN 60825 1 1994 A2 2001 The lasers are fully contained within the instrument structure and call for no special work area safety requirements except during service procedures These procedures are to be carried out only by BD Biosciences service personnel We recommend that you operate your BD LSR II flow cytometer only with the optics cover in place and the instrument lids closed When operated under these conditions the instrument poses no danger of exposure to hazardous laser radiation A Ar prevent injury do not modify the op
15. red 633 nm UV See Lasers on page 33 325 nm or 355 nm and violet 405 nm optics cover Shielding that houses the flow cell forward scatter FSC assembly see Detectors on page 35 and the excitation optics 32 BD LSR II User s Guide Lasers The BD LSR II flow cytometer has a fixed alignment 488 nm laser with the option of additional fixed alignment lasers Table 1 1 BD LSR II flow cytometer laser options Wavelength Power Warmup Laser Type Time Color mW min Standard Coherent Sapphire solid state 488 nm blue 20 30 JDS Uniphase 1344P helium 633 nm red 17 20 neon HeNe gas Coherent Radius 405 solid state 405 nm violet 25 15 Optional Lightwave Xcyte solid state 355 nm UV 20 30 OR Kimmon HeCd helium 325 nm UV 8 60 cadmium gas e The primary blue 488 nm laser Coherent Sapphire generates forward scatter FSC and side scatter SSC signals and four fluorescence signals e The optional red 633 nm laser JDS Uniphase 1344P generates two fluorescence signals e The optional violet 405 nm laser Coherent Radius 405 generates two fluorescence signals e The optional ultraviolet laser Lightwave Xcyte solid state 355 nm or Kimmon HeCd 325 nm generates two fluorescence signals M Tip To extend the life of the UV 325 nm HeCd laser turn on the instrument for at least 4 hours every week Chapter 1 Introduction 33 34 Filters Optical filters attenuate l
16. 100 150 200 FSC A Beads_001 Singlets PerCP positive 250 x 1 000 Beads_001 Singlets PE positive APC positive Tube Beads_001 Population BB all Events LA singlets HE FITC positive HII PE positive HI PerCP positive E APC positive Specimen Name FourColorBeads Tube Name Beads_001 FITC A Population Mean Events Parent Total 10 000 9 675 4 774 1 499 1 529 1 829 100 0 968 96 8 493 477 155 15 0 158 153 189 183 H Singlets 1 969 GB FITC positive 3 880 H FE positive 95 E FercF positive 106 arc positive 91 78 BD LSR II User s Guide Reusing the Analysis Global worksheets allow you to apply the same analysis to a series of recorded Tubes Once you define an analysis for a Tube you can use it to analyze the remaining Tubes in the Experiment After viewing the data you can print the analysis or save it to a Tube specific worksheet see Saving the Analysis 1 Move the Acquisition pointer to the Tube Beads_002 2 View the Beads_002 data on your analysis worksheet Adjust gates as needed Mi Tip Adjustments will apply to subsequent Tubes viewed on the worksheet You can avoid altering a global worksheet by saving an analysis to a Tube specific worksheet and making adjustments on the Tube specific worksheet 3 Print the analysis Saving the Analysis When an analysis is performed with a global worksheet the analysis is not saved with th
17. II instrument octagon and trigon arrays are shown in Table E 1 on page 164 BD LSR II User s Guide BD LSR II Workstation Acquisition analysis and most BD LSR II instrument functions are controlled by the BD LSR II workstation It includes a PC one or two monitors and a printer Your workstation is equipped with the following e a BD Biosciences validated Microsoft Windows operating system e BD FACSDiva software for data acquisition and analysis see the BD FACSDiva Software Reference Manual e software documentation including an online Help system Chapter 1 Introduction 37 38 BD LSR II User s Guide 2 Instrument Setup Starting the Flow Cytometer and Computer on page 40 Setting Up the Optical Filters and Mirrors on page 41 Preparing Sheath and Waste Containers on page 46 Preparing the Fluidics on page 49 Quality Control on page 51 39 Starting the Flow Cytometer and Computer 1 Turn on the system e Turn on the power to the flow cytometer Allow 60 minutes for lasers to warm up and stabilize A Failure to warm up and stabilize the lasers could affect sample data e Start up the BD LSR II workstation and log in to Windows M Tip You can turn on the power to the flow cytometer and the workstation in any order 2 Launch BD FACSDiva software by double clicking the shortcut on the desktop Log in to the software For details about user accounts in BD FACSDiva software see the BD FACSDiva Softw
18. J power receptacle ATTENTION Haute tension WASTE System waste tank Meaning Waste VISIBLE AND OR INVISIBLE LASER RADIATION WHEN REMOVED AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION DANGER RAYONNEMENT LASER VISIBLE ET OU INVISIBLE LORS DU RETRAIT EVITER L EXPOSITION OCULAIRE OU CUTANEE AU RAYONNEMENT DIRECT OU DIFFUSE B Meaning BD FACSFlow solution sheath Caution Handle biological substances with care Refer to manual for handling procedure DANGER Visible and or invisible laser radiation when removed Avoid eye or skin exposure to direct or scattered radiation None labels BD FACSFlow solution sheath port Near all removable covers and any place where the laser beam can emerge from the instrument Near cytometer BD FACSFlow solution sheath port BD LSR II User s Guide Limitations For research use only Not for use in diagnostic or therapeutic procedures BD Biosciences delivers software and workstations that are intended for running the instruments supplied by BD Biosciences It is the responsibility of the buyer user to ensure that all added electronic files including software and transport media are virus free If the workstation is used for Internet access or purposes other than those specified by BD Biosciences it is the buyer user s responsibility to install and maintain up to date virus protection software BD Bioscienc
19. M Tip Do not restart data recording during a calcium flux experiment Chapter 5 Calcium Flux 97 98 Setting Up the Experiment 1 Select Instrument gt Instrument Configuration and verify the current configuration Figure 3 1 on page 57 shows an Instrument Configuration dialog whose current configuration is the standard default configuration Verify that Indo 1 Violet and Indo 1 Blue appear in the current configuration For accurate data results the Instrument Configuration dialog box must reflect the physical layout of the BD LSR II octagons and trigons Modifications to the current configuration will not apply unless you click Set Configuration Select Edit gt User Preferences Verify that under the General tab all checkboxes are deselected see Figure 3 2 on page 58 See the BD FACSDiva Software Reference Manual for more information about the Instrument Configuration and User Preferences Create a new Experiment and rename it CalciumFlux With the CalciumFlux Experiment browser item selected verify in the Inspector frame that the Use global instrument settings checkbox is enabled see Figure 3 4 on page 60 Select the Instr Settings item of the CalciumFlux Experiment and use the Inspector frame to specify parameters and settings for your Experiment e With the Parameters tab delete all parameters except FSC SSC Indo 1 Blue and Indo 1 Violet Verify that the Log checkbox is deselected for all
20. Perform the system flush at least every 2 weeks AA Instrument hardware might be contaminated with biohazardous material Use 10 bleach to decontaminate the BD LSR II flow cytometer Flushing with 10 bleach is the only procedure recommended by BD Biosciences for decontaminating the instrument 1 Remove the sheath filter press the quick disconnects on both sides of the filter assembly remove the filter assembly connect the two fluid lines Mi Tip Do not run detergent bleach or ethanol through the sheath filter They can break down the filter paper within the filter body causing particles to escape into the sheath fluid possibly clogging the flow cell Chapter 6 Maintenance 111 10 11 12 13 Empty the sheath container and rinse it with DI water Fill the sheath container with at least 1 L of a 1 10 dilution of bleach or full strength BD FACS cleaning solution Empty the waste container if needed Open the roller clamp by the fluidics interconnect and drain the fluid into a beaker for 5 seconds Remove the DI water tube from the SIP Prime twice perform the following twice e Press the PRIME button on the fluidics control panel e When the STNDBY button lights amber press the PRIME button again Install a tube with 3 mL of a 1 10 dilution of bleach or full strength BD FACS cleaning solution on the SIP Press the RUN fluid control button run the cytometer on HI for 30 minutes Press the STND
21. Violet 6 Violet Indo 1 Blue B Indo 1 Violet A 9 DAPI A 9 APC c 10 Alexa Fluor 700 B 11 APC Cy7 A 12 upgrade previous detectors for instrument LSRII of detectors completed previous lasers for instrument LSRII of lasers completed upgrade configuration for instrument LSRII new lasers for instrument 6 Blue 0 Violet 2 UV 3 Red new fluorophores for instrument laser Blue of fluorophores completed new detectors for instrument laser Blue of fluorophores completed new fluorophores for instrument laser UY of fluorophores completed new detectors for instrument laser uv of fluorophores completed new fluorophores for instrument laser Red of fluorophores completed new detectors for instrument laser Red of fluorophores completed of lasers completed of new configuration complete ompleted Appendix G Instrument Configuration Application 201 5 Click Save Log to save a file containing the current text of the ICA main text view A Save As dialog is displayed Figure G 3 6 Choose a location and name for your log file then click Save Figure G 3 Select Configuration Save As El x Save in ICA 7 ce ConfigDatabase History E a D esktop aa My Computer My Network P File name UpgradeLog Pa Save as type ra files txt Cancel Zo 7 Click Exit to exit the program Restoring Your Default Configuration If you accidentally delete your BD FACS
22. blank none red trigon A 755 LP 780 60 APC Cy7 633 nm laser B 710 LP 730 45 Alexa Fluor 700 C 660 20 APC 186 BD LSR II User s Guide 6 Blue 6 Violet 2 UV 4 Red Configuration 6 Blue 6 Violet 2 UV 4 Red specifies blue violet and red octagons and a UV trigon Table F 8 shows the detectors filters and mirrors used in the default configuration The word blank indicates that a blank optical holder should be used instead of a mirror or filter A dash indicates that no slot exists for a mirror in that PMT position The 6 Blue 6 Violet 2 UV 4 Red maps are e 6 Color Blue Octagon Default Configuration Map on page 190 e 6 Color Violet Octagon Default Configuration Map on page 192 e 2 Color UV Trigon Default Configuration Map on page 193 e 4 Color Red Octagon Default Configuration Map on page 195 Table F 8 6 Blue 6 Violet 2 UV 4 Red default mirror and filter configuration Longpass Detector Array PMT Dichroic Bandpass Fluorochrome or Laser Detector Filter Scatter Parameter Mirror blue octagon A 755 LP 780 60 PE Cy7 488 nm laser B 685 LP 695 40 PerCP CyS 5 C 655 LP 660 20 PE Cy5 D 600 LP 610 20 PE Texas Red E 550 LP 575 26 PE F 505 LP 530 30 FITC Alexa Fluor 488 G blank 488 10 SSC H blank none Appendix F Optional Default Configurations 187 Table F 8 6 Blue 6 Violet 2 UV 4 Red default mirror and filter configuration continued
23. calculate compensation settings 3 Select Instrument gt Instrument Setup gt Calculate Compensation When the calculation is complete the Single Stained Setup dialog is displayed Enter a name for the compensation Setup and click OK Single Stained Setup Compensation calculation has completed successfully Name Jost 604jtm Mi Tip To help track compensation Setups include the Experiment name date or both in the Setup name The compensation Setup is linked to the MyExperiment instrument settings and subsequent acquisitions in MyExperiment are performed with the new compensation settings Chapter 3 Running Samples 69 Recording and Analyzing Data This section outlines some basic acquisition and analysis tasks using BD FACSDiva software The example shows data from two 4 color bead samples with the following fluorochromes e FITC e PE e PerCP e APC The procedure builds on the results obtained in the previous exercise Sample Optimization Using Instrument Setup on page 55 If you use a different sample type or if you have skipped the optimization exercise your software frame content and your data plots and statistics may deviate from those shown here Additionally you may need to modify some of the instructions in the procedure For additional details on completing some of the steps below see the BD FACSDiva Software Reference Manual Preparing the Workspace In this section you prepare y
24. instructions see Technical Assistance on page xvi 146 BD LSR II User s Guide Threshold The threshold is the level at which the system starts to measure signal pulses A threshold is defined for a specific detector signal The system continuously samples the digitized signal data and calculates pulse area height and width for all channels based on the time interval during which the threshold is exceeded Thresholds can also be set for more than one parameter and pulse measures are based on either of the following e intervals during which ALL signals exceed their threshold value e intervals during which ANY signal exceeds its threshold value Laser Controls Controls in the Laser tab of the Instrument frame are used to set the laser Delay Area Scaling and Window Extension values These parameters are set by BD Biosciences service personnel when the BD LSR II flow cytometer is installed and they rarely need to be changed Record and save these parameter values for future reference If needed see Appendix H for instructions on optimizing laser delay settings Do not otherwise change the settings in the Laser tab unless instructed to do so by BD Biosciences Changing the settings will affect your data Status Laser Name Area Scaling 1 0 00 1 00 2 22 00 1 00 3 40 80 1 00 4 61 90 1 00 Window Extension 10 000 Hl Instrument Connected Appendix A Technical Overview 147 148 BD LSR II User
25. it 116 BD LSR II User s Guide Remove the Old Filter 7 Place the instrument in STNDBY Disconnect the air line green Depressurize the sheath container by lifting the vent valve For a description of the vent valve see Figure 2 3 on page 46 Detach the cytometer fluid line from the filter assembly by squeezing the quick disconnect Detach the sheath container fluid line from the filter assembly by squeezing the quick disconnect Remove the vent line from the filter set aside Twist to remove Discard the used filter assembly Attach the New Filter 1 2 Connect the vent line to the filter Twist to attach Connect the sheath container fluid line to the filter assembly via the quick disconnect Attach the cytometer fluid line to the filter assembly via the quick disconnect Turn on the instrument to pressurize the sheath container Loosen the filter s vent cap to bleed off any air in the sheath filter Carefully tap the filter assembly to dislodge any air trapped in the filter element Loosen the filter s vent cap again to bleed off any air in the sheath filter Chapter 6 Maintenance 117 Changing the Bal Seal The sample injection tube Bal seal is a Teflon ring that forms a seal with the sample tube and ensures proper tube pressurization Over time this seal becomes worn or cracked and requires replacement Replacement is necessary if a proper seal is not formed when a sample tube is installed
26. nm laser B 685 LP 695 40 PerCP CyS 5 C 655 LP 660 20 PE Cy5 D 600 LP 610 20 PE Texas Red E 550 LP 575 26 PE F 505 LP 530 30 FITC Alexa Fluor 488 G blank 488 10 SSC H blank none UV trigon A 505 LP 530 30 Indo 1 Blue 325 nm or 355 nm laser B blank 450 50 Indo 1 Violet DAPI C blank none red trigon A 755 LP 780 60 APC Cy7 633 nm laser B 710 LP 730 45 Alexa Fluor 700 C 660 20 APC Appendix F Optional Default Configurations 179 6 Blue 2 Violet 2 UV 3 Red Configuration 6 Blue 2 Violet 2 UV 3 Red specifies a blue octagon and violet UV and red trigons Table F 4 shows the detectors filters and mirrors used in the default configuration The word blank indicates that a blank optical holder should be used instead of a mirror or filter A dash indicates that no slot exists for a mirror in that PMT position The 6 Blue 2 Violet 2 UV 3 Red maps are e 6 Color Blue Octagon Default Configuration Map on page 190 e 2 Color Violet Trigon Default Configuration Map on page 191 e 2 Color UV Trigon Default Configuration Map on page 193 e 3 Color Red Trigon Default Configuration Map on page 194 Table F 4 6 Blue 2 Violet 2 UV 3 Red default mirror and filter configuration Detector Array PMT on gpass Bandpass Fluorochrome or Dichroic i Laser Detector Filter Scatter Parameter Mirror blue octagon A 755 LP 780 60 PE Cy7 488 nm laser B 685 LP 695 40 PerCP CyS 5
27. on the SIP Mi Tip Indications that a proper seal has not formed include e the tube will not stay on the SIP without the tube support arm e the tube is installed RUN is pressed on the cytometer and the RUN button is orange not green To replace the Bal seal 1 Remove the outer droplet sleeve from the sample injection tube by turning the retainer counterclockwise Figure 6 6 Removing the outer sleeve Mi Tip Work carefully the outer sleeve can fall out as you loosen the retainer 118 BD LSR II User s Guide 2 Remove the Bal seal by gripping it between your thumb and index finger and pulling Figure 6 7 Figure 6 7 Removing the Bal seal 3 Install the new Bal seal spring side up Gently push the seal in place to seat it 4 Reinstall the retainer and outer sleeve over the sample injection tube Tighten the retainer just enough to hold it in place 5 Install a sample tube on the SIP to ensure that the outer sleeve has been properly installed If the sleeve hits the bottom of the tube loosen the retainer slightly and push the sleeve up as far as it will go Tighten the retainer Chapter 6 Maintenance 119 Changing the Sample Tube O Ring The sample tube O ring located within the retainer forms a seal that allows the droplet containment vacuum to function properly Replace the O ring when droplets form at the end of the sample injection tube while the vacuum is Operating AA Instrument hardware might be co
28. optics 56 components 32 cover 32 custom filter configurations DsRed 172 indo 1 171 PerCP or BD Cy Chrome 173 PE Texas Red 170 dichroic mirrors 34 141 filters 34 44 137 location 26 steering 32 35 optimization 55 calcium flux 97 calcium sample 101 data recording 93 for DNA 82 sample 55 ordering spare parts 160 O ring sample tube replacing 120 P parameters Time 97 PerCP and Stokes shift 136 PerCP filter configuration for 173 PE Texas Red filter configuration for 170 photodiode 35 photomultiplier tubes PMTs description 32 35 PMT voltages 55 population hierarchy 75 power requirements xix switch 26 priming fluidics system 51 propidium iodide PI cleaning after using 108 DNA QC 84 212 BD LSR II User s Guide pulse definition 144 measurements 145 processors 145 Q quality control QC about 51 log 149 particles 160 troubleshooting 131 R reconstructing default instrument configuration 43 recording calcium flux data 104 compensation Tubes 68 data 70 72 optimization 93 red laser 33 removing air bubbles filter 49 50 replacing Bal seal 118 battery 114 optical filters 44 sample tube O ring 120 sheath filter 116 117 requirements temperature and environmental xiv reusing analyses 79 running samples 53 S safety biological xx electrical xix general xix laser xvii symbols and labels xxi SAMPLE FINE ADJ knob 28 sample flow rate control buttons 28 sample injection port
29. parameters Verify that the A area checkbox is selected for Indo 1 Blue and Indo 1 Violet BD LSR II User s Guide Inspector ET sso Indo 1 Blue r indo 1 violet e With the Ratio tab click the Add button Select Indo 1 Violet A for the Numerator and Indo 1 Blue A for the Denominator 8 Inspector 6 Create a new Specimen in the CalciumFlux Experiment 7 Open the Specimen to access its Tube Rename the Tube CaF_001 and move the Acquisition pointer to CaF_001 Chapter 5 Calcium Flux 99 At this point your Experiment should look similar to EE calciumPlux HS Instr Settings ae Specimen_001 LT car_o01 8 Create a normal worksheet for the CaF_001 Tube with the following plots e FSC A vs SSC A dot plot e Indo 1 Blue A vs Indo 1 Violet A dot plot e Time vs Ratio Indo 1 Violet A Indo 1 Blue A dot plot 9 Select all plots Click the Title tab in the Inspector and specify displaying only the Tube and Population names in the plot titles Title Content IV Tube IV Populations a Specimen IO m Title Font sansSerit v Size 12 color BY 7 taic Bola 100 BD LSR II User s Guide 10 11 Create a Statistics view and display the mean for Indo 1 Blue A Indo 1 Violet A and the ratio parameter e Select Populations gt Create Statistics View e Right click on the Statistics view and select Edit S
30. s Guide Appendix B BD LSR II QC Log This sample quality control QC log can be photocopied or used as a guide in designing your own QC log 149 150 BD LSR II User s Guide s e1u 103e1ado 101 p eg 10118118 aWeN equiny jelas juawinsysu 1 15 Appendix B BD LSR II OC Log 152 BD LSR II User s Guide Appendix C Filter Templates You can use these templates to note your custom filter configurations for the BD LSR II instrument 153 154 BD LSR II User s Guide Experiment 156 BD LSR II User s Guide Trigon Template Laser Experiment Y Appendix C Filter Templates 157 158 BD LSR II User s Guide Appendix D Supplies and Consumables e Contact Information on page 160 e Consumables on page 160 159 Contact Information To order spare parts and consumables such as bulk fluids from BD Biosciences e within the US call 877 237 8995 prompt 2 2 4 e outside the US contact your local BD Biosciences customer service representative Worldwide contact information can be found at www bdbiosciences com Consumables QC Particles Immunocytometry Systems Particle Laser Supplier Catalog No e BD Calibrite 3 beads blue 488 nm BD Biosciences e 340486 unlabeled FITC Immunocytometry PE PerCP Systems e BD Calibrite APC beads e red 633 nm e 340487 e SPH
31. shown in this example is from a 4 color bead sample with the following fluorochromes e FITC e PE e PerCP e APC If you follow this procedure with a different bead sample or another sample type your software frame content and your data plots and statistics may deviate from those shown here Additionally you may need to modify some of the instructions in the procedure Verifying Instrument Configuration and User Preferences To obtain accurate data results the current BD FACSDiva software instrument configuration must reflect your BD LSR II instrument optics Verify the configuration and preferences before you create an Experiment 1 Select Instrument gt Instrument Configuration and verify the current configuration Figure 3 1 on page 57 shows an Instrument Configuration dialog whose Current Configuration is the standard default configuration BD LSR II User s Guide Figure 3 1 Instrument Configuration dialog Instrument Configuration FSC ssc FITC PE e E PE Cy7 Check that the configuration includes the parameters to be measured and that the channels correspond to the optical mirrors and filters For our bead sample the Instrument Configuration includes the following parameters FITC PE PerCP and APC Chapter 3 Running Samples 57 2 Select Edit gt User Preferences e Verify that the settings under the General tab match those displa
32. steps 2 and 3 with DI water 6 Set the fluid control to STNDBY Chapter 6 Maintenance 109 7 Place a tube containing no more than 1 mL of DI water on the SIP A tube with 1 mL of DI water should remain on the SIP to prevent salt deposits from forming in the injection tube This tube also catches back drips from the flow cell M Tip Do not leave more than 1 mL of water on the SIP When the BD LSR II flow cytometer is turned off or left in STNDBY mode a small amount of fluid will drip back into the sample tube If there is too much fluid in the tube it could overflow and affect instrument performance Daily Shutdown 1 Set the cytometer to SINDBY mode 2 Turn off the flow cytometer 3 Pull up on the sheath container vent valve to release pressure in the tank M Tip If the instrument will not be used for a week or longer perform a system flush see page 111 and leave the fluidics system filled with DI water to prevent saline crystals from clogging the fluidics 110 BD LSR II User s Guide Scheduled Maintenance Perform maintenance as indicated in Table 6 1 Table 6 1 Scheduled maintenance timetable Procedure Schedule Page System Flush once every 2 weeks page 111 Laser Maintenance once every week page 112 Waste Management System once every 2 weeks page 113 Maintenance System Flush An overall fluidics cleaning is required to remove debris and contaminants from the sheath tubing waste tubing and flow cell
33. tables list conventions used throughout this guide Table 1 lists the symbols that are used in this booklet or on safety labels 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 in material damage A data 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 M Tip Highlights features or hints that can save time and prevent difficulties 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 xii BD LSR II User s Guide BD LSR Il Documentation BD LSR II Online Help The online Help installed with your BD FACSDiva software contains the same text as that in the documents listed below enhanced with features like full text search and related topic links BD LSR II Online Help is accessed fro
34. the BD logo BD Calibrite BD FACS BD Cy Chrome BD FACSFlow BD Falcon and BD FACSDiva are trademarks of Becton Dickinson and Company Texas Red and Pacific Blue are trademarks and Alexa Fluor and Marina Blue are registered trademarks of Molecular Probes Inc Sapphire and Radius are trademarks and Coherent is a registered trademark of COHERENT INC ALPHA is a trademark of Omega Optical Inc SPHERO is a trademark of Spherotech Inc Xcyte is a trademark of Lightwave Electronics Modfit LT is a trademark of Verity Software House Inc Microsoft and Windows are registered trademarks of Microsoft Corporation Teflon is a registered trademark of E I du Pont de Nemours and Company JDS Uniphase is a trademark of JDS Uniphase Inc All other company and product names might be trademarks of the respective companies with which they are associated Patents BD LSR II is covered by one or more of the following US patents and foreign equivalents 4 745 285 4 844 610 and 6 014 904 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 BD FACS lysing solution US 4 654 312 4 902 613 and 5 098 849 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 equipm
35. time scale Signals are aligned with respect to the blue laser so the blue laser will have a 0 delay value and the red laser will have the largest delay 204 BD LSR II User s Guide Optimizing the Laser Delay Setting Laser Delay is set using BD FACSDiva software To optimize the delay for a given laser you acquire events from a sample with a fluorescence signal excited by that laser Follow the procedures in Chapter 3 Running Samples on page 53 for sample optimization and acquiring data 1 While acquiring data from your sample create a histogram plot to show the fluorescence signal excited by the laser whose delay is to be optimized 2 Inthe Acquisition Controls frame set the Events to Display to 500 evt 3 Select the Laser tab of the Instrument frame Figure H 2 Window Extension and Laser Delay values are displayed in psec Figure H 2 Laser tab of the Instrument frame Status Parameters Threshold Compensation Ratio Laser Blue 0 00 1 00 Violet 21 00 1 00 Uy 49 00 1 00 Red 66 90 1 00 Window Extension fi 0 000 St Fsc Area Scaling 1 00 At Instrument Connected 4 Set the Window Extension value to 0 psec Appendix H Setting Laser Delay 205 5 Set an initial Laser Delay value ONLY for the laser whose delay is to be optimized e If you are optimizing the violet laser set its delay to 20 psec e If you are optimizing the UV laser set its delay to 40 psec e If you are op
36. violet trigon none none none optional 405 nm violet laser UV trigon 450 LP Indo 1 Blue optional 325 nm UV laser or 355 nm UV laser 405 20 Indo 1 Violet red trigon none none none optional 633 nm red laser Appendix E Standard Default Configuration 169 PE Texas Red To use PE Texas Red replace the mirror and filter for the B PMT of the blue octagon as shown below 488 nm blue laser 170 BD LSR II User s Guide Indo 1 If you have a violet laser replace the mirror for the A PMT of the UV trigon as shown on the left below If you do not have a violet laser replace both the mirror for the A PMT and the filter for the B PMT of the UV trigon as shown on the right below 325 or 355 nm UV laser 325 or 355 nm UV laser with violet laser no violet laser Appendix E Standard Default Configuration 171 DsRed To use DsRed replace the filter for the C PMT of the blue octagon as shown below 488 nm blue laser 172 BD LSR II User s Guide PerCP or BD Cy Chrome Reagent To use PerCP or BD Cy Chrome reagent replace the mirror and filter for the B PMT of the blue octagon as shown below 488 nm blue laser Appendix E Standard Default Configuration 173 174 BD LSR II User s Guide Appendix F Optional Default Configurations The BD LSR II cytometer can be ordered with or upgraded to one of several laser and detector array options Each of these options comes with a
37. will slide out Figure 6 3 Opening drawer 2 Remove the drawer 3 Remove the battery from the drawer Figure 6 4 on page 115 4 Replace a new 9 volt battery into the drawer making sure that it is in the correct orientation Markings in the drawer will guide you 114 BD LSR II User s Guide Figure 6 4 Changing battery 5 Slide the drawer into the bracket until you feel a click 6 Test the new battery by following the procedure on page 113 Periodic Maintenance The following instrument components should be checked occasionally and cleaned as necessary The frequency will depend on how often the instrument is run Other components should be checked periodically for wear and replaced if necessary e Changing the Sheath Filter on page 116 e Changing the Bal Seal on page 118 e Changing the Sample Tube O Ring on page 120 Chapter 6 Maintenance 115 Changing the Sheath Filter The sheath filter Figure 6 5 connected to the top of the sheath container filters the sheath fluid as it comes from the sheath container Increased debris appearing in an FSC vs SSC plot can indicate that the sheath filter needs to be replaced We recommend changing the sheath filter assembly every 3 6 months Figure 6 5 Sheath filter cytometer fluid line quick disconnect vent cap vent line quick disconnect sheath container fluid line M Tip To avoid spraying sheath fluid depressurize the sheath container before opening
38. 0 UV 3 Red default mirror and filter configuration Longpass Detector Array PMT Dichroic Bandpass Fluorochrome or Laser Detector Filter Scatter Parameter Mirror blue octagon A 755 LP 780 60 PE Cy7 488 nm laser B 685 LP 695 40 PerCP CyS 5 C 655 LP 660 20 PE Cy5 D 600 LP 610 20 PE Texas Red E 550 LP 575 26 PE F 505 LP 530 30 FITC Alexa Fluor 488 G blank 488 10 SSC H blank none violet octagon A 630 LP 655 8 Qdot 655 405 nm laser B 595 LP 605 12 Qdot 605 C 575 LP 585 15 Qdot 585 D 545 LP 560 20 Qdot 565 E 475 LP 525 50 AmCyan Qdot 525 F blank 450 50 Pacific Blue G blank blank none H blank none red trigon A 755 LP 780 60 APC Cy7 633 nm laser B 710 LP 730 45 Alexa Fluor 700 C 660 20 APC 182 BD LSR II User s Guide 6 Blue 6 Violet 0 UV 4 Red Configuration 6 Blue 6 Violet 0 UV 4 Red specifies blue violet and red octagons Table F 6 shows the detectors filters and mirrors used in the default configuration The word blank indicates that a blank optical holder should be used instead of a mirror or filter A dash indicates that no slot exists for a mirror in that PMT position The 6 Blue 6 Violet 0 UV 4 Red maps are e 6 Color Blue Octagon Default Configuration Map on page 190 e 6 Color Violet Octagon Default Configuration Map on page 192 e 4 Color Red Octagon Default Configuration Map on page 195 Table F 6 6 Blue 6
39. 100 150 200 250 DAPI x 1 000 6 Use the Zoom In tool to magnify the area showing the singlets and doublets on the DAPI A vs DAPI W plot as shown in Figure 4 6 Figure 4 6 Doublet discrimination with zoomed plot 1 000 DAPLA 50 100 150 45139 150723 DAPI oe 1000 If you are unable to distinguish the singlet and doublet populations see Chapter 7 Troubleshooting 92 BD LSR II User s Guide 7 Draw a gate around the singlet CTN events 8 Adjust the event rate to approximately 200 events second with the SAMPLE FINE ADJ knob 9 Click Record to save the data 10 Print the worksheet 11 Remove the CTN tube from the cytometer and put the instrument in standby mode Optimization for Data Recording Optimize the instrument settings for the actual sample 1 Install the sample tube and adjust the FSC and SSC voltages to place the data events on scale 2 Adjust the DAPI voltage to place the singlet diploid events at about channel 50 x 103 After optimizing the instrument settings record data for each sample tube BD FACSDiva software does not include DNA analysis algorithms Export your data files for analysis in a third party application such as ModFit LT Chapter 4 DNA Analysis 93 94 BD LSR II User s Guide o Calcium Flux The following topics are covered in this chapter e Intracellular Calcium Concentration on page 96 e Calcium Flux Optimization on page 97 Recording Cal
40. 26 location 44 shown 36 template 157 tube support arm 30 Tubes compensation 61 U ungrounded receptacles xix unstained control 62 Use global instrument settings 60 User Preferences 58 84 98 dialog 58 UV laser 33 V violet laser 33 virus protection software xxiii voltages PMT adjusting 63 W waste container 31 46 alarm 31 capacity 31 emptying 48 window extension 205 workstation about 37 214 BD LSR II User s Guide
41. 5 holder 42 incident angle 45 minimum clear aperture 45 thickness 45 batch analysis 54 battery test 113 Battery Test switch shown 113 battery changing 114 BD Calibrite beads 131 BD FACS cleaning solution 109 BD FACS Instrument Configuration Application ICA 197 202 BD FACS rinsing solution 109 BD FACSDiva software See software BD FACSFlow solution 131 BD LSR II components 26 instrument optics 56 online Help xiii workstation 37 beads Calibrite 131 beam splitters See dichroic mirrors bi exponential scaling 54 biohazards xx 31 blank optical holders 42 bleach solution 109 blue laser 33 43 207 Browser toolbar buttons 59 buttons fluid control 28 sample flow rate control 28 C calcium flux about 96 Experiment 98 measuring 104 optimization 97 optimizing sample 101 Time parameter 97 calculating compensation 68 69 calf thymocyte nuclei CTN preparing 83 resolution 91 running 91 Calibrite beads 131 capacity waste container 31 CEN See chicken erythrocyte nuclei changing battery 114 chicken erythrocyte nuclei CEN optimization 84 preparing 83 running 89 Class I laser product xviii Coherent Radius 33 Sapphire 33 compensation calculating 68 69 theory 141 Tubes creating 61 values 55 components instrument shown 26 optical bench 32 computer system about 37 208 BD LSR II User s Guide configuration maps 165 containers sheath and waste 31 46 49 control stained 62 unstained 62 control pane
42. 8 nm laser 685 LP 695 40 PerCP CyS 5 655 LP 660 20 PE CyS 600 LP 610 20 PE Texas Red 550 LP 575 26 PE 505 LP 530 30 FITC Alexa Fluor 488 blank 488 10 SSC blank none red trigon 755 LP 780 60 APC Cy7 633 nm laser 710 LP 730 45 Alexa Fluor 700 OO me gt oO aol ale oO oO we 660 20 APC 176 BD LSR II User s Guide 6 Blue 2 Violet 0 UV 3 Red Configuration 6 Blue 2 Violet 0 UV 3 Red specifies a blue octagon and violet and red trigons Table F 2 shows the detectors filters and mirrors used in the default configuration The word blank indicates that a blank optical holder should be used instead of a mirror or filter A dash indicates that no slot exists for a mirror in that PMT position The 6 Blue 2 Violet 0 UV 3 Red maps are e 6 Color Blue Octagon Default Configuration Map on page 190 e 2 Color Violet Trigon Default Configuration Map on page 191 e 3 Color Red Trigon Default Configuration Map on page 194 Table F 2 6 Blue 2 Violet 0 UV 3 Red default mirror and filter configuration Detector Array PMT en gpass Bandpass Fluorochrome or Dichroic Laser Detector Mirror Filter Scatter Parameter blue octagon 755 LP 780 60 PE Cy7 488 nm laser 685 LP 695 40 PerCP CyS 5 655 LP 660 20 PE Cy5 600 LP 610 20 PE Texas Red 550 LP 575 26 PE 505 LP 530 30 FITC Alexa Fluor 488 blank 488 10 SSC blank none
43. A 1 000 7 Display only the Lymphocyte population in the remaining two dot plots Select the two plots right click inside one of the plots and choose Show Populations gt Lymphocytes 8 Adjust the Indo 1 Violet A and the Indo 1 Blue A voltages to optimize the signal The signal when displayed in the plot should resemble that of Figure 5 3 102 BD LSR II User s Guide Figure 5 3 Optimized Indo 1 signal 250 150 200 100 2 Q a z a T a e Nos j 3 100 150 200 250 Indo 1 Violet A 61 000 9 Set the mean of the ratio parameter to about 50 000 e Select the CaF_001 Tube in the Browser and click the Ratio tab in the Inspector e Adjust the value in the Scaling field a higher percentage will raise the mean until the mean of the ratio parameter is about 50 000 use Statistics View Your time vs ratio plot should resemble o Indo 1 VIOLETI A Inds 1 BLUE 260 x 1 000 Chapter 5 Calcium Flux 103 Recording Calcium Flux Data To record data for a calcium flux experiment 1 4 In the Acquisition Controls frame change the Events to Display to 50 000 events Verify that the unstimulated sample is still running The event rate is displayed in the Acquisition Status frame Use the cytometer s SAMPLE FINE ADJ knob to adjust the event rate to about 200 events second In the Acquisition Controls frame click Record Keep the instrument in R
44. A CD on an available drive If the installer setup program does not start automatically launch setup exe from the CD root folder After the setup runs the InstallShield Wizard will lead you through several installation screens On the Setup Type screen select Complete installation When the installation is complete click Finish and eject the CD Re installing ICA If ICA is already installed on your computer the InstallShield Wizard welcome screen asks you if you want to modify repair or remove the program To re install ICA first remove it then rerun the installer Uninstalling ICA To remove ICA from your computer you can either e Run the ICA installer and remove the program as described above e Use your operating system s Add or Remove Programs control panel 198 BD LSR II User s Guide Updating Your Default Configuration When you upgrade your BD LSR II instrument configuration your BD FACSDiva software default instrument configuration must be updated 1 2 Launch ICA from the program list of your Start menu The ICA activity window is displayed Figure G 1 on page 200 shows the ICA activity window as it would appear after step 3 below With the Select Instrument pull down specify LSRIT The Select Configuration scroll view lists the available BD LSR II instrument configurations Select the BD LSR II instrument configuration that matches your configuration upgrade The main text area displays
45. BD LSR Il User s Guide AW Y http www bdbiosciences com Part No 338639 Rev A October 2004 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 O 2004 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
46. BY fluid control button and depressurize the sheath container by lifting the pressure relief valve cap Repeat steps 2 through 10 with BD FACS rinsing solution in place of the bleach solution Repeat steps 2 through 10 with DI water in place of the bleach solution Replace the sheath filter and refill the sheath container with sheath fluid Laser Maintenance To extend the life of the HeCd laser turn on the instrument for 3 4 hours at least once per week 112 BD LSR II User s Guide Waste Management System Maintenance The waste management system for the BD LSR II instrument has an alarm powered by a 9 volt battery that must be tested and changed regularly to ensure its continued operation Test the battery every 2 weeks after you flush the system Change the battery as needed Testing the Battery and Alarm 1 Locate the Battery Test switch on the waste container bracket Figure 6 2 Figure 6 2 Battery Test switch 2 Toggle the switch If the battery and the alarm are working properly you should hear buzzing If you do not hear any sound change the battery as described in the following section 3 Release the switch Chapter 6 Maintenance 113 Changing the Battery You will need the following supplies to change the battery e small flat head screwdriver e 9 volt battery Perform the following steps 1 Insert the tip of a flat head screwdriver into the slot as shown in Figure 6 3 and pry it out A small drawer
47. Diva software default instrument configuration the BD FACS Instrument Configuration Application ICA can be used to restore it Follow the instructions in the previous section for a configuration upgrade except select your non upgraded instrument configuration from the configurations list When you click Upgrade your previous default instrument configuration will be restored 202 BD LSR II User s Guide Appendix H Setting Laser Delay This appendix outlines a procedure for optimizing the laser delay settings in a multiple laser system 203 Laser Delay Sample interrogation takes place within the cuvette flow cell Laser light is directed through a series of prisms that focus multiple lasers on the event stream at different positions This allows optimal detection of fluorescent signal from each laser with minimal cross contamination from the other beams In the BD LSR II four laser system the blue laser intercepts the stream first followed by the violet UV and red lasers Because the laser signals are spatially separated there is a slight delay between the detection of each laser s signal Figure H 1 Figure H 1 Signal separation over time event intercepts red laser event intercepts UV laser red event intercepts violet laser event intercepts blue laser UV violet ooo blue The Laser Delay factor in BD FACSDiva software is used to realign the signals so they can be measured and displayed on the same
48. ERO e all e BD Biosciences e 559123 Rainbow Pharmingen Calibration Particles 8 peak e SPHERO Ultra e all e Spherotech Inc e URFP Rainbow 30 2 Fluorescent Particles single peak DNA QC Particles kit blue 488 nm BD Biosciences 349523 160 BD LSR II User s Guide Reagents Reagent Supplier Catalog No BD FACSFlow sheath fluid BD Biosciences 340398 US and Latin America 342003 Europe Monoclonal antibodies BD Biosciences A BD FACS lysing solution BD Biosciences 349202 BD FACS rinsing solution BD Biosciences 340346 BD FACS cleaning solution BD Biosciences 340345 Dyes and fluorochromes BD Biosciences e Clontech e Pharmingen Molecular Probes Sigma Chlorine bleach 5 sodium hypochlorite Clorox or other major supplier to ensure that the bleach is at the correct concentration and free of particulate matter a Refer to the BD Biosciences Product Catalog or the BD Biosciences website http www bdbiosciences com Appendix D Supplies and Consumables 161 Equipment Equipment Item Supplier Catalog No Bal seal BD Biosciences 343509 O ring sample tube 343615 Sheath filter assembly 344678 BD Falcon polystyrene test tubes BD Labware 352052 12 x 75 mm 352054 352058 162 BD LSR II User s Guide Appendix E Standard Default Configuration The standard default configuration for a BD LSR II cytometer specifies d
49. LSR II User s Guide 4 DNA Analysis The following topics are covered in this chapter Criteria for DNA Experiments on page 82 CEN Optimization on page 84 CTN Resolution on page 91 Optimization for Data Recording on page 93 81 Criteria for DNA Experiments 82 To support DNA experiments a flow cytometer must provide e ability to resolve histogram peaks populations e linearity of the DNA fluorescence signal e ability to distinguish singlets from aggregates While obtaining good peak resolution for DNA also depends on proper sample preparation optimization of instrument optics and fluidics is critical The ability of a flow cytometer to resolve peaks can be assessed by measuring the CV of a reference particle the lower the CV the better the resolution Linearity is also critical for DNA experiments To assess the linearity of DNA data the pulse area signal is used to measure the amount of DNA fluorescence detected from cells or nuclei On an instrument with good linearity the doublet peak should be located at twice the mean channel of the singlet peak Figure 4 1 Figure 4 1 Area signal and DNA fluorescence DNA QC KitCEN oa E 532 3 5 Doublets fi 50 100 150 200 250 10739 164 DAPI A 001 000 BD LSR II User s Guide Doublet discrimination or the ability to distinguish singlets from aggregates is also important for DNA experiments Since doublets of Go G cells have the same amoun
50. See page 120 Outer sleeve not seated in the retainer 1 Loosen the retainer Figure 6 6 on page 118 2 Push the outer sleeve up into the retainer until seated 3 Tighten the retainer Outer sleeve not on the sample injection tube Replace the outer sleeve 1 Loosen the retainer 2 Slide the outer sleeve over the sample injection tube until it is seated 3 Tighten the retainer Waste line pinched preventing proper aspiration Check the waste line Waste tank full Empty the waste tank Sample tube not fitting on SIP Sample tube other than BD Falcon tubes used Use BD Falcon 12 x 75 mm sample tubes See page 162 Worn Bal seal Replace the Bal seal See page 118 Rapid sample aspiration Support arm to the side Place the support arm under the sample tube Droplet containment module failing Call your service representative 122 BD LSR II User s Guide Instrument Troubleshooting continued Observation No events in acquisition display and green RUN button Possible Causes Threshold not set to correct parameter usually FSC Recommended Solutions Set the threshold to the correct parameter for your application Threshold level too high Lower the threshold level PMT voltage for threshold parameter set too low Set the PMT voltage higher for the threshold parameter Refer to the BD FACSDiva Software Reference Manual for instructions
51. Tube When recording has completed remove the tube from the cytometer Perform steps 2 through 4 below for each stained control tube in the order in which they appear in the Browser in the Compensation Controls item To correctly calculate compensation all Tubes must be recorded with the same PMT voltage settings If you change a PMT voltage you must restart with a new set of Compensation Controls 2 Install the stained control tube onto the cytometer 3 In the Acquisition Controls frame click the Next button Click the Acquire button to start acquisition Click Record to record data 4 When recording has completed remove the tube from the cytometer Calculating Compensation Settings In this section you adjust a gate for the fluorescence positive population of each stained control Tube Then you calculate compensation for your Instrument Setup sample Perform steps 1 and 2 below for each stained control Tube 1 Click the corresponding worksheet tab to display the Tube plots 2 Instrument Setup automatically creates a P2 Snap To interval gate on the fluorescence histogram Adjust the gate to encompass the fluorescence positive population only BD LSR II User s Guide An example of an adjusted P2 gate is shown in Figure 3 13 Figure 3 13 Gating the positive population PE Stained Control S Q Q 2 s a a 150 200 260 FSC A x 1 000 After all of the fluorescence positive gates have been adjusted you can
52. UN mode as you perform steps 5 through 7 below Do not stop recording The unstimulated sample data must be maintained If recording is stopped ensure that the subsequent data is appended to the unstimulated sample data 5 When approximately 10 000 events have been recorded remove the unstimulated sample tube from the cytometer Add the stimulus to the tube and mix thoroughly Reinstall the tube on the SIP After a few seconds the Ca concentration begins to increase on the Time vs Ratio plot Figure 5 4 on page 105 104 BD LSR II User s Guide Figure 5 4 Cellular response to stimulus over time CaF_001 Lymphocytes rm A ES e f LILL 150 T TT 250 x 1 000 unstimulated stimulus sample added 8 When the sample has stopped reacting to the stimulus click the Acquire button in the Acquisition Controls frame to stop acquisition and data recording Mi Tip To verify the end of the stimulus response change the Events to Display back to 500 events and check that the mean of the ratio parameter has stabilized 9 Remove the tube from the cytometer 10 Clean the fluidics system with 10 bleach for 5 minutes and then with DI water for 5 minutes M Tip Residual stimulus must be removed from the system Any remaining stimulus would activate cells in subsequent samples To run additional samples 11 Install the next sample tube on the SIP 12 In the Acquisition Contr
53. Violet 0 UV 4 Red default mirror and filter configuration Detector Array PMT en gpass Bandpass Fluorochrome or Dichroic Laser Detector Mirror Filter Scatter Parameter blue octagon 755 LP 780 60 PE Cy7 488 nm laser 685 LP 695 40 PerCP CyS 5 655 LP 660 20 PE Cy5 600 LP 610 20 PE Texas Red 550 LP 575 26 PE 505 LP 530 30 FITC Alexa Fluor 488 blank 488 10 SSC TO mM m0 0 wu gt blank none Appendix F Optional Default Configurations 183 Table F 6 6 Blue 6 Violet 0 UV 4 Red default mirror and filter configuration continued Longpass Detector Array PMT Dichroic Bandpass Fluorochrome or Laser Detector Filter Scatter Parameter Mirror violet octagon A 630 LP 655 8 Qdot 655 405 nm laser B 595 LP 605 12 Qdot 605 C 575 LP 585 15 Qdot 585 D 545 LP 560 20 Qdot 565 E 475 LP 525 50 AmCyan Qdot 525 F blank 450 50 Pacific Blue G blank blank none H blank none red octagon A 755 LP 780 60 APC Cy7 633 nm laser B 710 LP 730 45 Alexa Fluor 700 C 675 LP 685 35 Alexa Fluor 680 D 660 20 APC E blank blank none F blank blank none G blank blank none H blank none 184 BD LSR II User s Guide 6 Blue 6 Violet 2 UV 3 Red Configuration 6 Blue 6 Violet 2 UV 3 Red specifies blue and violet octagons and UV and red trigons Table F 7 shows the detectors filters and mirrors used in the default configuratio
54. ainer Ensure that the sheath container lid is tight and all connectors are secure Hypertonic buffers or fixative Replace the buffers and fixative Excessive amount of debris in display Threshold level too low Increase the threshold level Sheath filter dirty Replace the filter See Changing the Sheath Filter on page 116 Flow cell dirty Flush the system See System Flush on page 111 Dead cells or debris in sample Examine the sample under a microscope Sample contaminated Re stain the sample ensure tube is clean Stock sheath fluid contaminated Rinse the sheath container with DI water then fill with sheath fluid from another or new lot bulk container Chapter 7 Troubleshooting 129 Instrument Troubleshooting continued Observation High CV Possible Causes Air bubble in sheath filter or flow cell Recommended Solutions Purge the filter See Removing Air Bubbles on page 49 Sample flow rate set too high Set the sample flow rate lower Air leak at sheath container Ensure that the sheath container lid is tight and all connectors are secure Flow cell dirty Flush the system See System Flush on page 111 Poor sample preparation Repeat sample preparation Sample not diluted in same fluid as sheath fluid Dilute the sample in the same fluid as you are using for sheath Old or contaminated quality control QC
55. an of the Singlet population The mean ratio should be 2 00 0 05 If you do not achieve a ratio between 1 95 2 05 contact BD Customer Support e Copy the population means and CVs and the calculated linearity result into your QC log Remove the CEN sample from the cytometer 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 Width plot On the Area vs Width plot singlets are distinguished from doublets by the Width measurement singlets have a smaller Width measurement Discriminating the singlets from the aggregates enhances the accuracy of cell cycle analysis Running CTN 1 2 3 4 On the BD LSR II cytometer control panel press RUN and LO Install the CTN sample tube on the cytometer Adjust the event rate to approximately 500 events second Click the Next button in the Acquisition Controls frame 48 DNA A new Tube is created in the DNA OC Kit specimen i p Y Change the name of the Tube to CTN ECEN U CTN The Next button duplicates the CEN Tube and Analysis objects The new plots and Statistics view appear below the previous objects on the worksheet Acquisition for the CTN Tube starts automatically Chapter 4 DNA Analysis 91 5 Adjust the DAPI voltage to place the first peak at approximately channel 50 x 103 on the DAPLA axis Figure 4 5 Unresolved singlets and doublets in unzoomed plot 50
56. ar to that in Figure 3 8 on page 64 4 Click the Threshold tab in the Instrument frame Adjust the FSC Threshold to remove most of the debris without clipping the singlet population Chapter 3 Running Samples 63 64 To gate the population of interest bead singlets 5 Instrument Setup automatically creates a P1 Snap To polygon gate on the FSC A vs SSC A plot of the Unstained Control worksheet Adjust the P1 gate as needed to encompass the singlet population only Figure 3 8 See the BD FACSDiva Software Reference Manual for more information about adjusting Snap To polygon gate boundaries Figure 3 8 P1 gate adjusted to singlet population Unstained Control ze 2 aoa 2 g ya Tao Y an 2 os 2 o 50 150 200 250 4972 260046 FSC A x 1 000 6 Right click on the gate and select Apply to All Compensation Tubes Figure 3 9 Figure 3 9 Apply to All Compensation Tubes Unstained Control SSC A x 1 000 60 190 150 200 250 Apply to All Comprjpsation Controls Recalculate Delete Delete 100 150 200 250 FSC A x 1 000 BD LSR II User s Guide To adjust fluorescence PMT voltages 7 Display log decade gridlines on the fluorescence histograms e Select all fluorescence histograms on the Unstained Control worksheet e Inthe Inspector select the Show Grid checkbox Figure 3 10 Figure 3 10 Inspector for fluorescent plots Inspector
57. are Reference Manual 3 Check the BD FACSDiva software Instrument frame to ensure that the cytometer is connected to the workstation The cytometer connects automatically While connecting the message Instrument Connecting is displayed in the frame footer When connection completes the footer message changes to Instrument Connected Figure 2 1 on page 41 If the footer message Instrument Disconnected appears see Troubleshooting on page 121 40 BD LSR II User s Guide Figure 2 1 Instrument Connected message Instrument x Instrument Connected Setting Up the Optical Filters and Mirrors Before you run samples you should set up the optical filters The figure below shows the location of the detector arrays beneath the instrument covers of the BD LSR II flow cytometer Each detector array is labeled with its laser source red trigon UV trigon violet trigon blue octagon Chapter 2 Instrument Setup 41 42 Filter and Mirror Configurations Each PMT except the last PMT in every detector array has two slots in front of it e The slot closest to the PMT holds a bandpass filter holder e The slot furthest from the PMT holds a longpass dichroic mirror holder The last PMT in every detector array PMT H in the octagon and PMT C in all trigons does not have a mirror slot Optical Holders Filters and Mirrors Optical holders house filters and mirrors Your instrument includes several bla
58. ce eee eens Preparing the Waste Container 00 cece cece eee eee ees Preparing the Fluidics 1 0 0 ccc cc cece eee e teenies Removing Air Bubbles ooooooooooonooroonooooooo Priming the Fluidics ici id a deta ee ea bes Quality Controls occa cea a Ca eee as BD LSR II User s Guide 25 26 26 26 27 27 28 28 28 29 31 32 33 34 35 37 39 40 41 42 44 45 46 46 48 49 49 51 51 Chapter 3 Running Samples Sample Optimization Using Instrument Setup o ooooooocoooomoo o Verifying Instrument Configuration and User Preferences Creating an Experiment Adjusting Voltages and Threshold Setting o oooooomom Recording Compensation Tubes 0 00 e cece cece cece eee Calculating Compensation Settings 06 cece cece eee Recording and Analyzing Data Preparing the Workspace Recording Data Analyzing Data Reusing the Analysis Saving the Analysis Chapter 4 DNA Analysis Criteria for DNA Experiments DNA Setup How to Use DAPI with DNA QC keene ees How to Use PI with DNA QC CEN Optimization Preparing the Workspace Running CEN CIN Resolution Running CTN Optimization for Data Recording Contents 53 55 56 59 63 68 68 70 70 72 75 79 79 81 82 83 83 84 84 84 89 91 91 93 vil Chapter 5 Ca
59. cium Flux Data on page 104 95 Intracellular Calcium Concentration 96 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 Ca 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 5 1 A break in data occurs when the stimulus is added to the sample tube The increase in
60. default configuration These optional default configurations are described in e 6 Blue 0 Violet 0 UV 3 Red Configuration on page 176 e 6 Blue 2 Violet 0 UV 3 Red Configuration on page 177 e 6 Blue 0 Violet 2 UV 3 Red Configuration on page 178 e 6 Blue 2 Violet 2 UV 3 Red Configuration on page 180 e 6 Blue 6 Violet 0 UV 3 Red Configuration on page 181 e 6 Blue 6 Violet 0 UV 4 Red Configuration on page 183 e 6 Blue 6 Violet 2 UV 3 Red Configuration on page 185 e 6 Blue 6 Violet 2 UV 4 Red Configuration on page 187 Octagon and trigon maps for the optional configurations are found in Optional Default Configuration Trigon and Octagon Maps on page 189 175 6 Blue 0 Violet 0 UV 3 Red Configuration 6 Blue 0 Violet 0 UV 3 Red specifies a blue octagon and a red trigon Table F 1 shows the detectors filters and mirrors used in the default configuration The word blank indicates that a blank optical holder should be used instead of a mirror or filter A dash indicates that no slot exists for a mirror in that PMT position The 6 Blue 0 Violet 0 UV 3 Red maps are e 6 Color Blue Octagon Default Configuration Map on page 190 e 3 Color Red Trigon Default Configuration Map on page 194 Table F 1 6 Blue 0 Violet 0 UV 3 Red default mirror and filter configuration Detector Array PMT Lon gpass Bandpass Fluorochrome or Dichroic Laser Detector Mirror Filter Scatter Parameter blue octagon 755 LP 780 60 PE Cy7 48
61. dth calculates aa x 64000 Appendix A Technical Overview 145 Digital Electronics BD LSR II flow cytometer electronics digitize the signal intensity produced by a detector The digitized data is stored in memory and further processed by the electronics to calculate e pulse height area and width e compensation e parameter ratios These results are transferred to your workstation computer for further processing by BD FACSDiva software For more information about digital theory refer to the Digital Theory appendix in the BD FACSDiva Software Reference Manual BD FACSDiva Option White Paper For an in depth discussion of digital electronics visit our website at http www bdbiosciences com immunocytometry_systems and download the BD FACSDiva Option White Paper To download e From the main menu on the Immunocytometry Systems welcome page select Products gt Literature which takes you to the Literature in Print page e On the Literature in Print page click the link Download Print from the Literature Library which takes you to the Literature Library page e On the Literature Library page at the top left under the View Literature Type heading click Application Notes Technical White Papers then click the View link on the line with BD FACS FACSDiVa Option White Paper If you have difficulty downloading this white paper from the web contact your local BD Biosciences technical support representative or supplier for updated
62. e BD FACSDiva Software Ouick Start Guide For additional details see your BD FACSDiva software online help and the BD FACSDiva Software Reference Manual Related Topics Additional topics related to the material of this chapter are covered in the BD FACSDiva Software Quick Start Guide and the BD FACSDiva Software Reference Manual These include e Batch analysis e Bi exponential scaling 54 BD LSR II User s Guide Sample Optimization Using Instrument Setup Before you record data for a sample the following should be optimized for the sample type and fluorochromes used e PMT voltages e threshold setting e compensation values This section describes how to optimize samples using the Instrument Setup feature of BD FACSDiva software In particular Instrument Setup automatically calculates compensation settings If you choose to perform compensation manually not all of the instructions given below will apply For detailed instructions on completing some of the steps below see the BD FACSDiva Software Reference Manual Sample optimization consists of several main steps to be performed in the order listed below Each of the steps is explained in greater detail in the sections that follow 1 Verify instrument configuration and user preferences Create an Experiment Adjust voltages and the threshold setting 2 3 4 Record the Compensation Tubes 5 Calculate compensation settings Chapter 3 Running Samples 55 S6 The data
63. e Changing Optical Filters or Mirrors on page 44 Laser not functioning Verify laser malfunction by changing the threshold to an alternative laser while running appropriate QC particles If not successful contact BD Biosciences High event rate Air bubble in sheath filter or flow cell Remove the air bubble See Removing Air Bubbles on page 49 Threshold level too low Increase the threshold level Refer to the BD FACSDiva Software Reference Manual for instructions PMT voltage for threshold parameter set too high Set the PMT voltage lower for the threshold parameter Refer to the BD FACSDiva Software Reference Manual for instructions Sample too concentrated Dilute the sample Sample flow rate set on HI Set the sample flow rate to MED or LO 126 BD LSR II User s Guide Instrument Troubleshooting continued Observation Possible Causes Recommended Solutions Low event rate Threshold level too high Lower the threshold level Refer to the BD FACSDiva Software Reference Manual for instructions PMT voltage for threshold Set the PMT voltage higher for the parameter set too low threshold parameter Refer to the BD FACSDiva Software Reference Manual for instructions Sample not adequately mixed Mix the sample to suspend cells Sample too dilute Concentrate the sample If the flow rate setting is not critical to the application set the flow rate switch to MED or HI
64. e Tube Mi Tip If you define your analysis on a global worksheet before recording data you can specify to automatically save the analysis after recording data This option is set in User Preferences To save a copy of the analysis of Beads_001 with that Tube 1 Expand the MyDataAnalysis global worksheet item in the Browser 2 Right click on its Analysis and select Copy 3 Click the Template tool B1 on the Worksheet toolbar to switch to the normal worksheet view Chapter 3 Running Samples 79 4 Select Worksheet gt New Worksheet to create a new normal worksheet Click on the new worksheet and then use the Inspector to rename it Beads_001_Analysis e Select the Beads_001 item in the Browser Right click the Beads_001 Tube icon and select Paste EHO MyFolder Z MyExperiment mS Instr Settings HB Global Worksheets a MyData A MyDataAnalysis Lad Analysis E FourColorBeads Te Beads_001 Beads_002 E Compensation Controls 8S Instr Settings ad Unstained Control EJE Cut Ctrl x i Copy Ctrl C Delete Delete The Analysis objects from the MyDataAnalysis global worksheet are copied to the Beads_001_Analysis normal worksheet You can view the analysis by double clicking the Beads_001 Tube icon in the Browser M Tip You can apply the global worksheet analysis to multiple Tubes on a single normal worksheet by selecting more than one Tube before you paste the Analysis 80 BD
65. e are found in Appendix F Changing Optical Filters or Mirrors A A Follow the precautions outlined in Laser Safety on page xvii while changing optical filters or mirrors 1 Lift the appropriate instrument cover e The octagon array is located under the right instrument cover e The three trigon arrays are located under the left instrument cover M Tip To open the left instrument cover you must first open the right cover and the side door 2 Remove the appropriate filter holder or mirror holder 3 Replace it with the new filter holder or mirror holder M Tip The filters and mirrors fit easily into the optical holders in only one way 4 Close the instrument cover s BD LSR II User s Guide Custom Configurations See Custom Configurations in Appendix E for specifications of some common custom filter and mirror configurations The custom filters and mirrors used in these configurations are included with your spares kit Filter and Mirror Specifications Table 2 1 Longpass dichroic mirrors in octagon or trigon Specification Measurement diameter 0 625 in 0 000 0 005 minimum clear aperture 0 562 in incident angle 11 25 thickness 0 125 in 0 005 in Table 2 2 Bandpass filters in octagon or trigon Specification Measurement diameter 1 00 in 0 010 in minimum clear aperture 0 85 in incident angle 0 thickness 0 12 0 35 in Chapter 2 Instrument Setup 45 Pr
66. e flow cell The RUN button is green when the sample tube is on and the support arm is centered When the tube support arm is moved left or right to remove a sample tube the instrument switches to an automatic standby status to conserve sheath fluid the RUN button changes to orange e STNDBY standby stops fluid flow to conserve sheath fluid When you leave the instrument for more than a few minutes place a tube containing 1 mL of deionized DI water on the sample injection port SIP and press STNDBY BD LSR II User s Guide e PRIME prepares the fluidics system by draining and filling the flow cell with sheath fluid The fluid flow initially stops and pressure is reversed to force fluid out of the flow cell and into the waste container After a preset time the flow cell fills with sheath fluid at a controlled rate to prevent bubble formation or entrapment At completion the instrument switches to STNDBY mode Sample Injection Port The sample injection port SIP is where the sample tube is installed The SIP includes the sample injection tube and the tube support arm Samples are introduced through a stainless steel injection tube equipped with an outer droplet containment sleeve The sleeve works in conjunction with a vacuum pump to eliminate droplet formation of sheath fluid as it backflushes from the sample injection tube Figure 1 3 Sample injection port SIP Bal seal outer sleeve tube stop sample injection t
67. e is required contact your local BD Biosciences technical support representative or supplier When contacting BD Biosciences have the following information available e product name part number and serial number e version of BD FACSDiva software you are using e any error messages e details of recent system performance For instrument support from within the US call 877 232 8995 prompt 2 2 For support from within Canada call 888 259 0187 Customers outside the US and Canada contact your local BD representative or distributor xvi BD LSR II User s Guide Safety and Limitations The BD LSR II flow cytometer is equipped with safety features for your protection Operate the instrument only as directed in the BD LSR IT User s Guide and the BD LSR II Safety and Limitations booklet Keep this information available for future reference and for new users Laser Safety Lasers or laser systems emit intense coherent electromagnetic radiation that has the potential of causing irreparable damage to human skin and eyes The main hazard of laser radiation is direct or indirect exposure of the eye to thermal radiation from the visible and near infrared spectral regions 325 1400 nm Direct eye contact can cause corneal burns retinal burns or both and possible blindness There are other potentially serious hazards in other spectral regions Excessive ultraviolet exposure produces an intolerance to light photophobia accompanied
68. e optimization 59 extra filters and mirrors 169 F FACSFlow solution 131 Falcon tubes 122 filters bandpass 34 default configurations 164 optical bandpass 34 139 changing 44 dichroic 34 discriminating 140 extra 169 longpass 34 138 shortpass 34 139 specifications 45 theory 137 sheath replacing 116 117 FITC and Stokes shift 136 fixed alignment lasers 26 flow cell 134 draining 51 flow rate control buttons 28 fluid control buttons 28 Index 209 fluidics 134 cleaning 108 description 28 134 flow rate control 28 flushing system 111 priming 51 removing air bubbles 49 run mode 28 sample injection port SIP 29 30 fluorescence 136 emission 144 fluorochromes 141 emission spectra 137 filter configurations and 164 169 role in light emission 136 which measured 43 forward scatter FSC 32 33 135 FSC and SSC voltages adjusting 63 FSC threshold adjusting 63 G gate boundaries adjusting 64 gating data 75 global worksheet 75 gridlines log decade 65 H handles instrument 27 32 hazards biological xx 31 electrical xix laser xvii Hoechst 108 hydrodynamic focusing 134 210 BD LSR II User s Guide ICA 197 202 installing 198 uninstalling 198 immunophenotyping 134 analysis 75 data 71 Experiment 70 Include separate unstained control tube well checkbox 61 indo 1 filter configuration for 171 instrument components shown 26 configuration 43 covers and door 26 44 dimensions xiv
69. ed gray and the outer is blue BD LSR II User s Guide Figure 1 5 Dichroic filter types in octagon array bandpass filters longpass dichroic mirrors The steering optics and filters mounted on the BD LSR II instrument are listed in Table E 1 on page 164 See page 137 for a more detailed explanation of how filters work in the BD LSR II flow cytometer Detectors Light signals are generated as particles pass through the laser beam in a fluid stream When these optical signals reach a detector electrical pulses are created that are then processed by the electronics system There are two types of signal detectors in the BD LSR II flow cytometer the photodiode and the photomultiplier tubes or PMTs Figure 1 6 on page 36 A photodiode is less sensitive to light signals than the PMTs Therefore a photodiode is used to detect the stronger forward scatter signal PMTs are used to detect the weaker signals generated by side scatter and all fluorescence channels These signals are amplified by applying a voltage to the PMTs As the voltage is increased the detector sensitivity increases resulting in increased Chapter 1 Introduction 35 36 signal As the voltage is decreased the detector sensitivity decreases resulting in decreased signal Detector voltages are adjusted in BD FACSDiva software Figure 1 6 PMT type detectors in trigon array PMT PMT The default locations of specific detectors and filters within BD LSR
70. efault Configurations 189 6 Color Blue Octagon Default Configuration Map 488 nm blue laser 19 BD LSR II User s Guide 2 Color Violet Trigon Default Configuration Map 405 nm violet laser Appendix F Optional Default Configurations 191 6 Color Violet Octagon Default Configuration Map 405 nm violet laser 192 BD LSR II User s Guide 2 Color UV Trigon Default Configuration Map 325 or 355 nm UV laser opv Appendix F Optional Default Configurations 193 3 Color Red Trigon Default Configuration Map 633 nm red laser NO Alexa Fluor 700 Wa A E DS 194 BD LSR II User s Guide 4 Color Red Octagon Default Configuration Map 633 nm red laser Appendix F Optional Default Configurations 195 19 BD LSR II User s Guide Appendix G Instrument Configuration Application The BD FACS Instrument Configuration Application ICA is a utility distributed with your BD LSR II instrument If you upgrade your configuration ICA must be used to update your BD FACSDiva software default instrument configuration database object You can also use ICA to restore an accidentally deleted default configuration This appendix consists of e Installing ICA on page 198 e Updating Your Default Configuration on page 199 e Restoring Your Default Configuration on page 202 197 Installing ICA To install the BD FACS Instrument Configuration Application Mount your IC
71. ens and materials coming into contact with them are considered biohazardous Handle as if capable of transmitting infection Dispose of waste using proper precautions and in accordance with local regulations Never pipette by mouth Wear suitable protective clothing eyewear and gloves Figure 2 4 Waste container waste tubing from cytometer level sensor line 1 Disconnect the orange waste tubing and the black sensor line from the waste tank Keep the lid on the waste tank until you are ready to empty it 2 Empty the waste container AA WARNING The waste container contents might be biohazardous Treat contents with bleach 10 of total volume Dispose of waste using proper precautions and in accordance with local regulations Wear suitable protective clothing eyewear and gloves 48 BD LSR II User s Guide 3 Add approximately 1 L of bleach to the waste container and close it 4 Reconnect the orange waste tubing and make sure that it is not kinked 5 Reconnect the level sensor line black Preparing the Fluidics Next you should make sure the fluidics system is ready This section covers the following preparations e Removing Air Bubbles below e Priming the Fluidics on page 51 Removing Air Bubbles Trapped bubbles can occasionally dislodge and pass through the flow cell resulting in inaccurate data NOTICE Do not vigorously shake bend or rattle the sheath filter or you might damage it 1 Check the
72. ent NOTICE 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 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 334717 Rev A 12 02 Initial release 338639 Rev A 10 04 Second release Contents About This Guide Conventions oooooooo ooo mono ooo BD LSR II Documentation BD LSR II Online Help Printed Documentation 05 BD FACSDiva Software Help Menu BD Biosciences Website Download Technical Assistance o ooooooooooo o o Safety and Limi
73. eparing Sheath and Waste Containers Vv Tip Check the fluid levels in the sheath and waste containers every time you use the instrument This ensures that you will not run out of sheath fluid during an experiment and that the waste container will not become too full Preparing the Sheath Container Figure 2 3 Sheath container clamp knob air line vent valve fill point sheath tank cytometer fluid line filter assembly 46 BD LSR II User s Guide Make sure that the flow cytometer is in STNDBY mode Disconnect the air line green from the sheath container see Figure 2 3 on page 46 Depressurize the sheath container by pulling up on its vent valve Remove the sheath container lid Unscrew the clamp knob and lift Add 6 L of sheath fluid such as BD FACSFlow solution to the sheath container NOTICE Do not fill the sheath tank to its maximum capacity 8 L When a full tank is pressurized erratic instrument performance can result M Tip For calcium flux experiments use a sheath fluid that does not contain preservatives For example use 1X phosphate buffered saline PBS Close the sheath container lid Reconnect the air line green Make sure that the lid is finger tight and that the cytometer fluid line blue is not kinked M Tip Inspect the sheath container periodically as sheath fluid can cause corrosion Chapter 2 Instrument Setup 47 Preparing the Waste Container AA All biological specim
74. es Before opening the sheath container 1 Put the instrument in STNDBY mode 2 Disconnect the air line green 3 Depressurize the sheath container by lifting its vent cap Waste Container The waste container has a capacity of 10 L An alarm sounds when the container becomes full AA To avoid leakage of biohazardous waste put the instrument in STNDBY mode before disconnecting the waste container AA The waste container contents might be biohazardous Treat contents with bleach 10 of total volume Dispose of waste with proper precautions in accordance with local regulations Wear suitable protective clothing eyewear and gloves A The waste container is heavy when full When emptying it use good body mechanics to prevent injury Chapter 1 Introduction 31 Optics Figure 1 4 shows the optical bench components of the BD LSR II instrument Figure 1 4 Optical bench components engineering model red 633 nm laser photomultiplier tube PMT optional UV 325 or 355 nm laser optional violet 405 nm laser optional i steering optics photomultiplier tube PMT photomultiplier tubes PMTs Devices that convert optical signals into electrical signals see Detectors on page 35 octagon Array of PMTs and filters that can detect up to eight signals see Figure 1 5 on page 35 trigon Array of PMTs and filters that can detect up to three signals see Figure 1 6 on page 36 lasers blue 488 nm
75. es does not make any warranty with respect to the workstation remaining virus free after installation BD Biosciences is not liable for any claims BD Biosciences is not liable for any claims related to or resulting from buyer user s failure to install and maintain virus protection Safety and Limitations xxiii xxiv BD LSR II User s Guide Introduction The following topics are covered in this chapter e Overview on page 26 e Fluidics on page 28 e Optics on page 32 e BD LSR II Workstation on page 37 25 Overview The BD LSR IT is an air cooled multi laser benchtop flow cytometer with the ability to acquire parameters for a large number of colors It uses fixed alignment lasers that transmit light reflected by mirrors through a flow cell to user configurable octagon and trigon detector arrays These detectors collect and translate fluorescence signals into electronic signals Instrument electronics convert these signals into digital data Components Figure 1 1 BD LSR II flow cytometer left cover side door ath right cover fluidics interconnects control panel instrument handle power switch Power Switch The power switch is located on the lower right side of the BD LSR II instrument as shown in Figure 1 1 26 BD LSR II User s Guide Control Panel The control panel contains fluidics controls Figure 1 2 For a description of the controls see e Sample Flow Rate Control on page 28 e Fluid Con
76. es with the BD DNA QC Particles kit instead of DAPI To do so prepare the CEN and CTN samples as described in the kit instructions CEN Optimization 84 Use the following procedure to set up BD FACSDiva software for a DNA experiment that uses DAPI as the DNA staining dye If you are using another sample type modify the steps accordingly If you are using PI substitute PI for DAPI throughout the procedure Preparing the Workspace 1 Select Instrument gt Instrument Configuration and verify the current configuration Figure 3 1 on page 57 shows an Instrument Configuration dialog whose current configuration is the standard default configuration Verify that DAPI appears in the current configuration For accurate data results the Instrument Configuration dialog box must reflect the physical layout of the BD LSR II octagons and trigons Modifications to the current configuration will not apply unless you click Set Configuration 2 Select Edit gt User Preferences Verify that under the General tab all checkboxes are deselected see Figure 3 2 on page 58 See the BD FACSDiva Software Reference Manual for more information about the Instrument Configuration and User Preferences 3 Create a new Experiment and rename it DNA BD LSR II User s Guide 4 With the DNA Experiment browser item selected verify in the Inspector frame that the Use global instrument settings checkbox is enabled see Figure 3 4 on page 60 5 Select t
77. etectors filters and mirrors for one to four lasers The BD LSR II cytometer can also be ordered with one of several optional configurations The optional default configurations are specified in Appendix F 163 Standard 4 Blue 2 Violet 2 UV 2 Red Configuration Specification The standard configuration specifies a blue octagon and violet UV and red trigons Table E 1 shows the detectors filters and mirrors used in the standard default configuration The word blank indicates that a blank optical holder should be used instead of a mirror or filter A dash indicates that no slot exists for a mirror in that PMT position Table E 1 Default BD LSR II flow cytometer filter configuration Detector Array PMT Lon gpass Bandpass Fluorochrome or Dichroic e Laser Detector E Filter Scatter Parameter Mirror blue octagon A 735 LP 780 60 PE Cy7 488 nm laser B 685 LP 695 40 PerCP CyS 5 C 550 LP 575 26 PE PI D 505 LP 530 30 FITC FP E blank 488 10 SSC F blank blank none G blank blank none H blank none violet trigon A 505 LP 525 50 AmCyan 405 nm laser B blank 440 40 Pacific Blue C blank none UV trigon A 505 LP 530 30 Indo 1 Blue 325 nm or 355 nm laser B blank 450 50 Indo 1 Violet DAPI C blank none 164 BD LSR II User s Guide Table E 1 Default BD LSR II flow cytometer filter configuration Detector Array PMT con gpass Bandpass F
78. excited electronic state In returning to their ground state the dyes release energy most of which is emitted as light This light emission is known as fluorescence Fluorescence is always a longer wavelength lower energy photon than the excitation wavelength The difference between the excitation wavelength and the emission wavelength is known as the Stokes shift Some fluorescent compounds such as PerCP exhibit a large Stokes shift absorbing blue light 488 nm and emitting red light 675 nm while other fluorochromes such as FITC have a smaller Stokes shift absorbing blue light and emitting green light 530 nm The emission spectra for some commonly used fluorochromes are shown in Figure A 3 on page 137 136 BD LSR II User s Guide 100 Alexa Fluor 430 Cascade Blue APC PerCP PerCP Cy5 5 PE Cy7 normalized intensity 400 500 600 700 800 wavelength nm Figure A 3 Emission spectra of commonly used fluorochromes Optical Filters Optical filters modify the spectral distribution of light scatter and fluorescence directed to the detectors When photons encounter an optical filter they are either transmitted absorbed or reflected Figure A 4 Figure A 4 Effect of an optical filter on incident photons photons absorbed photons photons transmitted reflected Su Appendix A Technical Overview 137 138 Even though an optical filter is rated at its 50 transmission point the filter passes or lets throug
79. h a minimal amount of light outside of this indicated rating The slope of an optical filter transmission curve indicates filter performance A relatively steep slope indicates a high performance high quality optical filter that provides deep attenuation of out of band wavelengths A less steep slope indicates that more light outside the rated bandwidth is being transmitted Two kinds of filters are used on the BD LSR II flow cytometer e longpass LP e bandpass BP including discriminating filters DF and ALPHA filters AF A third filter type the shortpass SP is not recommended but can be used in some custom configurations LP BP and SP filters are referred to as dichroic filters Longpass Filter LP filters pass wavelengths longer than the filter rating For example a 500 LP filter permits wavelengths longer than 500 nm to pass through it and either absorbs or reflects wavelengths shorter than 500 nm longpass 480 520 100 4 460 500 540 transmission T T 450 500 550 wavelength nm BD LSR II User s Guide Shortpass Filter An SP filter has the opposite properties of a longpass filter An SP filter passes light with a shorter wavelength than the filter rating shortpass 480 520 460 500 540 transmission 450 500 550 wavelength nm Bandpass Filter A BP filter transmits a relatively narrow range or band of light Bandpass filters are typically designated by two numbe
80. he Instr Settings item of the DNA Experiment and use the Inspector frame to specify the parameters and threshold setting for your Experiment e Click the Parameters tab in the Inspector frame e Delete all parameters except FSC SSC and DAPI e Verify that the Log checkbox is deselected for all parameters e For DAPI verify that the A area checkbox is selected and select the W width checkbox for DAPI Inspector xj Instr Settings Parameters Threshold Ratio Compensation Parameter Chapter 4 DNA Analysis 85 e Click the Threshold tab and change the threshold parameter to DAPI verify that the threshold value is set to 5 000 Parameters Threshold Ratio Compensation or And Parameter 6 Create a new Specimen in the DNA Experiment and rename it DNA QC Kit 7 Open the Specimen to access its Tube Rename the Tube CEN and move the Acquisition pointer to CEN Your Experiment should look similar to the figure below E DNA 8 Instr Settings E A DNA QC Kit U cen 86 BD LSR II User s Guide 8 Click the Threshold tab and change the threshold parameter to DAPI verify that the threshold value is set to 5 000 x Parameters Threshold Ratio Compensation or And Parameter Print 9 Create a normal worksheet for the CEN Tube with the following plots e FSC A vs SSC A dot plot e DAPI A vs DAPI W dot plot e DAPI A histogram
81. ight or help direct it to the appropriate detectors The BD LSR TI instrument uses dichroic filters Dichroic filters transmit light of a specific wavelength while reflecting other wavelengths The name and spectral characteristics of each filter appear on its holder There are three types of dichroic filters e Shortpass SP filters transmit wavelengths that are shorter than the specified value e Longpass LP filters transmit wavelengths that are longer than the specified value e Bandpass BP filters pass a narrow spectral band of light by combining the characteristics of shortpass filters longpass filters and absorbing layers Discriminating filters DF and ALPHA filters AF are types of bandpass filters When dichroic filters are used as steering optics to direct different color light signals to different detectors they are called dichroic mirrors or beam splitters e Shortpass dichroic mirrors transmit shorter wavelengths of light to one detector while reflecting longer wavelengths to a different detector e Longpass dichroic mirrors transmit longer wavelengths to one detector while reflecting shorter wavelengths to a different detector The BD LSR II instrument octagon and trigon detector arrays use dichroic longpass mirrors on their inner rings and bandpass filters on their outer rings However you can customize the arrays with other types of filters and mirrors In Figure 1 5 on page 35 the inner ring is color
82. ings checkbox Inspector xi Experiment Keywords Name uyExperiment Owner administrator Modified fa 104 4 45 21 PM Log Decades for Plots 4 Log Decades 5 Log Decades IV Use global instrument settings 5 Specify the parameters for Instrument Setup e Inthe Browser select the Instr Settings of MyExperiment e Click the Parameters tab in the Inspector frame Delete any unnecessary parameters To delete a parameter select it by clicking the selection button to the left of the parameter name and then click the Delete button Figure 3 5 on page 61 e Verify that Log is deselected for the FSC and SSC parameters and is selected for each fluorescence parameter e Verify that A area is selected for every parameter and that H height and W width are deselected for every parameter BD LSR II User s Guide Figure 3 5 Deleting parameters for four color bead optimization Inspector xl Instr Settings Parameters Threshold Ratio Compensation a aja al gt E K 6 Select Instrument gt Instrument Setup gt Create Compensation Controls The Create Compensation Controls dialog is displayed Figure 3 6 e Verify that the Include separate unstained control tube well checkbox is selected and that the compensation control tube list matches the fluorescent parameters selected in step 5 above e For this bead example you do not need to provide no
83. is provides a standard for monitoring instrument performance Instrument QC consists of e running QC samples e recording parameter means and CVs QC data should be analyzed for trends involving 10 or more runs By keeping the instrument settings and QC sample constant changes in the recorded means and CVs will indicate how much instrument performance varies over time QC results are affected by laser and fluidics performance We strongly recommend following the laser and fluidics maintenance procedures in Chapter 6 Chapter 2 Instrument Setup 51 See QC Particles on page 160 for a list of acceptable QC beads A sample QC log is provided in Appendix B It can be photocopied or used as a guide in designing your own QC log 52 BD LSR II User s Guide 3 Running Samples This chapter describes procedures that use BD FACSDiva software to record and analyze sample data e Sample Optimization Using Instrument Setup on page 55 e Recording and Analyzing Data on page 70 53 Before You Begin Before attempting to perform the procedures in this chapter you should be familiar with e BD LSR I instrument startup setup and QC procedures see Chapter 2 Instrument Setup on page 39 BD FACSDiva software concepts workspace components instrument and acquisition controls tools for data analysis An excellent way to learn the BD FACSDiva software concepts required by this chapter is to perform the tutorial exercises in th
84. l instrument 27 controls compensation 61 single stained 56 70 cord damaged xix covers instrument 26 Create Compensation Controls 61 dialog 62 Create Statistics view 75 creating Acquisition Templates 70 Analysis objects 75 compensation Tubes 61 CTN See calf thymocyte nuclei custom configurations 169 cuvette flow cell 204 CV troubleshooting 130 Cy Chrome filter configuration for 173 D DAPI and DNA 83 cleaning after using 108 data analyzing 70 75 gating 75 recording 70 72 calcium flux 104 optimization 93 debris excessive 129 removing 63 default configuration restoring 202 Default global worksheet checkbox 58 default instrument configuration deleting 43 reconstructing 43 delay laser 204 detectors photodiode 35 photomultiplier tubes PMTs 35 sensitivity 36 dichroic filters 34 mirrors 34 141 digital data 26 discriminating filters DFs 140 DNA Experiment criteria 82 flow rate for analysis 134 QC Particles kit 83 using DAPI 83 using PI 83 verifying linearity 82 doublet discrimination 83 89 droplet containment system 30 troubleshooting 122 DsRed filter configuration for 172 E electrical requirements xiv safety xix electronics 144 emission duration 144 environmental requirements xiv event rate erratic 128 high 126 low 127 zero 123 125 excessive debris 129 excitation wavelength 136 Experiments calcium flux 98 DNA linearity 82 immunophenotyping 70 placement in folders 59 sampl
85. lcium Flux 95 Intracellular Calcium Concentration oo oooomomororormomormoo 96 Calcium Flux Optimization 0 ccc cece eee ence eens 97 Using the Time Parameter 0 0 e cece cece cece eee e eens 97 Setting Up the Experiment 0 0 c eee cece eee nens 98 Optimizing for Calcium Flux 0 cece cece eee eens 101 Recording Calcium Flux Data 0 cee ccc cece enn eee enees 104 Chapter 6 Maintenance 107 Daily Cleaning and Shutdown 0c c eee cece een eens 108 Daily Fluidics Cleaning 2 0 0 0 cece cc eee eee eee enes 108 Daily Shutdown 2 1 0 0 cece cece nono 110 Scheduled Maintenance 0 cece o 111 System Flush Gi icc A Be SR Ee a 111 Laser Maintenance ai ou ees Wyte dees 112 Waste Management System Maintenance 0 e eee eee 113 Perro di Maintenance ndee aa eve eee wed neds MaMa ee whee 115 Changing the Sheath Filter 02 0 cee cece eens 116 Changing the Bal Seal 0 ec ce eee eens 118 Changing the Sample Tube O Ring 00 cece cece eens 120 Chapter 7 Troubleshooting 121 Instrument Troubleshooting 0c e eee eens 122 viii BD LSR II User s Guide Appendix A Technical Overview Fluidics 20 A A E A aoa Optical Filters a a A A ede A Compensation Theory 0 ce cece eee eee ence eee e eens Electronics esgere Net hella ty ela ee ale ee Ens OS Pale eae Gs lees Pulse Measurements 2 4 otee aed MER eA RR ales ada Digital E
86. lectronics oooooooooooomorrrorromrrrroro s BD FACSDiva Option White Paper o ooooooocoomomommmm Enreshold sa EENT A ii Aad Bh dls Laser Controls IA dr ai Appendix B BD LSR Il QC Log Appendix C Filter Templates Octagon Template sin tenia is tr abe bya bees Trigon Template sovena A NAR As Pele Appendix D Supplies and Consumables Contact Information 0 cee cece nora Consumables aa Mae Paes ee Pasa el ek ee OG Particles a E A E RES e e a Sud o ets le a do ate Equipment suscita A a iii Contents 133 134 135 135 136 137 141 144 145 146 146 147 147 149 153 155 157 159 160 160 160 161 162 Appendix E Standard Default Configuration Standard 4 Blue 2 Violet 2 UV 2 Red Configuration Specification Custom Configurations Octagon and Trigon Maps Appendix F Optional Default Configurations 6 Blue 0 Violet 0 UV 3 Red Configuration oooooooomomomomo or 6 Blue 2 Violet 0 UV 3 Red Configuration 0 000 e eee eee eee 6 Blue 0 Violet 2 UV 3 Red Configuration o oooooomomomom or 6 Blue 2 Violet 2 UV 3 Red Configuration 0 000 sesen eee ee 6 Blue 6 Violet 0 UV 3 Red Configuration 0 0c cece e ences 6 Blue 6 Violet 0 UV 4 Red Configuration oooooooomomomom or 6 Blue 6 Violet 2 UV 3 Red Configuration ooooooomomomomo or 6 Blue 6 Violet 2 UV 4 Red Configuration 02 00 senres eeuse Optional Default Configurati
87. luorochrome or Dichroic E Laser Detector Filter Scatter Parameter Mirror red trigon A 735 LP 780 60 APC Cy7 633 nm laser B blank 660 20 APC C blank none Octagon and Trigon Maps This section contains maps of the default octagon and trigon mirror and filter configurations If a slot is filled with a filter or mirror an identifying number appears in that position on the configuration map If a slot is filled with a blank optical holder that position on the configuration map is unlabeled Appendix E Standard Default Configuration 165 Figure E 1 Standard default configuration blue octagon 488 nm blue laser 166 BD LSR II User s Guide Figure E 2 Standard default configuration red and violet trigons 633 nm red laser 405 nm violet laser O APC My a Appendix E Standard Default Configuration 167 Figure E 3 Standard default configuration UV trigon 325 or 355 nm UV laser O 168 BD LSR II User s Guide Custom Configurations This section specifies some common custom filter and mirror configurations The custom filters and mirrors used in these configurations are contained in the BD LSR TI instrument spares kit Extra Filters and Mirrors Instrument Spares Kit Detector Array Laser Mirrors Filters Fluorochrome blue octagon 600 LP 610 20 PE Texas Red standard 488 nm blue laser 635 LP 670 14 PerCP BD Cy Chrome reagent 585 42 DsRed
88. m the Help menu of BD FACSDiva software Help content is organized by topic and subsections which are available as links in the Contents tab BD LSR II Online Help topics are compiled from the following sources e BD LSR II User s Guide e BD FACSDiva Software Reference Manual BD High Throughput Sampler User s Guide Printed Documentation A printed copy of the following documents is distributed with the BD LSR II flow cytometer e BD LSR II User s Guide The BD LSR II User s Guide describes the procedures necessary to operate and maintain your BD LSR II flow cytometer Because many instrument functions are controlled by BD FACSDiva software this guide also contains information about software features required for basic instrument setup and operation e BD LSR II Safety and Limitations booklet The BD LSR II Safety and Limitations booklet discusses the safety features of the BD LSR II flow cytometer It lists precautions for the cytometer s laser electrical and biological hazards and states limitations of use About This Guide xiii xiv BD LSR II Facility Requirement Guide The BD LSR II Facility Requirement Guide contains specifications for instrument weight and size temperature and other environmental requirements electrical requirements BD FACSDiva Software Quick Start Guide The BD FACSDiva Software Quick Start Guide contains tutorial exercises that familiarize you with key software procedures and concepts
89. me of the FITC emission appears in the PE detector This can be seen in a dot plot of FITC vs PE Figure A 7 Figure A 7 Theoretical display of FITC vs PE without compensation FITC positive PE population unstained particles FITC This FITC spillover in the PE detector is to be corrected as indicated by the arrow in Figure A 7 Using the Compensation tab of the Instrument frame of BD FACSDiva software adjust the PE FITC spectral overlap value BD LSR II User s Guide Compensation is optimal when the positive and negative FITC populations have the same means or medians in the PE parameter statistics see Figure A 8 Figure A 8 FITC spillover optimally compensated out of the PE parameter FITC positive PE population unstained particles A means match FITC Once fluorescence compensation has been set for any sample the compensation setting remains valid for a subsequent dim or bright sample because compensation subtracts a percentage of the fluorescence intensity Figure A 9 illustrates this principle Although the signals differ in intensity the percentage of the FITC spillover into the PE detector remains constant Figure A 9 Two FITC signals of different intensity FITC PE different intensity FITC signals same proportion or percentage of spectral overlap in PE channel normalized intensity 500 nm 550 nm 00 nm 850 nm 700 nm Appendix A Technical Overview 143 Electronics
90. n The word blank indicates that a blank optical holder should be used instead of a mirror or filter A dash indicates that no slot exists for a mirror in that PMT position The 6 Blue 6 Violet 2 UV 3 Red maps are e 6 Color Blue Octagon Default Configuration Map on page 190 e 6 Color Violet Octagon Default Configuration Map on page 192 e 2 Color UV Trigon Default Configuration Map on page 193 e 3 Color Red Trigon Default Configuration Map on page 194 Table F 7 6 Blue 6 Violet 2 UV 3 Red default mirror and filter configuration Detector Array PMT con gpass Bandpass Fluorochrome or Dichroic Laser Detector z Filter Scatter Parameter Mirror blue octagon A 755 LP 780 60 PE Cy7 488 nm laser B 685 LP 695 40 PerCP Cy5 5 C 655 LP 660 20 PE CyS D 600 LP 610 20 PE Texas Red E 550 LP 575 26 PE F 505 LP 530 30 FITC Alexa Fluor 488 G blank 488 10 SSC H blank none Appendix F Optional Default Configurations 185 Table F 7 6 Blue 6 Violet 2 UV 3 Red default mirror and filter configuration continued violet octagon A 630 LP 655 8 Qdot 655 405 nm laser B 595 LP 605 12 Qdot 605 G 575 LP 585 15 Qdot 585 D 545 LP 560 20 Qdot 565 E 475 LP 525 50 AmCyan Qdot 525 F blank 450 50 Pacific Blue G blank blank none H blank none UV trigon A 505 LP 530 30 Indo 1 Blue 325 nm or F 355 nm laser B blank 450 50 Indo 1 Violet DAPI C
91. n generic tube labels e Click OK to create the control Tubes Chapter 3 Running Samples 61 Figure 3 6 Create Compensation Controls dialog Create Compensation Controls Compensation Controls are added to the Experiment along with a single stained control Tube for each parameter in the Experiment and an unstained control Tube Figure 3 7 Worksheets containing appropriate plots and gates are added for each compensation Tube Figure 3 7 Compensation Tubes E e Compensation Controls 25 Instr Settings U unstained Control U FITC Stained Control UJ PE Stained Control U PerCP Stained Control U APC Stained Control 62 BD LSR II User s Guide Adjusting Voltages and Threshold Setting In this section you use the unstained control tube to e adjust FSC and SSC voltages and FSC threshold e gate the population of interest bead singlets e adjust fluorescence PMT voltages To adjust scatter voltages and the threshold setting 1 Install the unstained control tube on the cytometer 2 Click the Current Tube pointer of the Unstained Control Tube it becomes green Click the Acquire button in the Acquisition Controls frame to begin acquisition 3 Adjust the FSC and SSC voltages to position the singlet bead population for gating e Click the Parameters tab in the Instrument frame e Use the up and down arrows or drag the voltage sliders to adjust the voltage settings When adjusted your data should look simil
92. ng the threshold to an alternative laser while running appropriate QC particles If not successful contact BD Biosciences 124 BD LSR II User s Guide Instrument Troubleshooting continued Observation No events in acquisition display and orange RUN button Possible Causes RUN not activated Recommended Solutions Press the RUN button Sample tube not installed or not properly seated Install the sample tube correctly on the SIP Sample tube cracked Replace the sample tube Sheath container not pressurized e Ensure that the sheath container lid and all connectors are securely seated e Inspect the O ring and replace if necessary See Changing the Sample Tube O Ring on page 120 Bal seal worn Replace the Bal seal See page 118 Air leak at sheath container Ensure that the sheath container lid and all connectors are securely seated Sheath container empty Fill the sheath container Air in sheath filter Purge the filter See Removing Air Bubbles on page 49 Chapter 7 Troubleshooting 125 Instrument Troubleshooting continued Observation No fluorescent signal Possible Causes Incorrect fluorochrome assignment Recommended Solutions Make sure the Instrument Configuration in the software matches the optical filters in the instrument Wrong filter installed Make sure the appropriate filter is installed for each fluorochrome Se
93. nk empty optical holders see Figure 2 2 Figure 2 2 Blank optical holders filters and mirrors bandpass filter longpass dichroic mirror loose 695 40 empty empty blank optical holder filter slot blank optical holder mirror slot BD LSR II User s Guide M Tip To ensure data integrity do not leave any slots unfilled in a detector array when you are using the associated laser Always use a blank optical holder Default Configurations Each BD LSR II instrument has a default filter and mirror configuration The standard default configuration is specified for an instrument with an octagon detector array for the 488 nm blue laser and trigon detector arrays for any optional lasers Appendix E gives a detailed description of the standard default configuration also called the 4 Blue 2 Violet 2 UV 2 Red default configuration Additional default configurations are specified for instruments with non standard detector arrays For example there is a 6 Blue 6 Violet 2 UV 3 Red default configuration for an instrument that has octagon detector arrays for both the 488 nm blue laser and the 405 nm violet laser and trigon detector arrays for both the UV laser and the 633 nm red laser Detailed descriptions of the additional default configurations are found in Appendix F BD FACSDiva Instrument Configuration When you acquire data using BD FACSDiva software you specify an instrument configuration The instrument config
94. ntaminated with biohazardous material Wear suitable protective clothing eyewear and gloves whenever cleaning the instrument or replacing parts 1 Remove the outer droplet sleeve from the sample injection tube Turn the retainer counterclockwise and pull the outer sleeve from the retainer see Figure 6 6 on page 118 2 Invert the retainer and allow the O ring to fall onto the benchtop If the O ring does not fall out initially tap the retainer on the benchtop to dislodge the O ring 3 Drop the new O ring into the retainer Make sure the O ring is seated properly in the bottom of the retainer 4 Reinstall the retainer and the outer sleeve 5 Install a sample tube on the SIP to ensure that the outer sleeve has been properly installed If the sleeve hits the bottom of the tube loosen the retainer slightly and push the sleeve up as far as it will go Tighten the retainer 120 BD LSR II User s Guide 7 Troubleshooting The tips in this section are designed to help you troubleshoot your experiments Additional troubleshooting information can be found in the BD FACSDiva Software Reference Manual If additional assistance is required contact your local BD Biosciences technical support representative See Technical Assistance on page xvi 121 Instrument Troubleshooting Observation Droplet containment vacuum not functioning Possible Causes Worn O ring in retainer Recommended Solutions Replace the O ring
95. ols frame click the Next button to create a new Tube Rename the new Tube appropriately 13 Repeat steps 3 through 10 Chapter 5 Calcium Flux 105 BD FACSDiva software does not include Calcium flux analysis algorithms Export your data files for analysis in a suitable third party application Figure 5 5 Calcium flux data points over time CaF_001 Lymphocytes resolution ly 2 l co at E uu 250 x 1 000 Ratic Ingo 161 106 BD LSR II User s Guide 6 Maintenance Daily Cleaning and Shutdown on page 108 Scheduled Maintenance on page 111 Periodic Maintenance on page 115 107 The BD LSR II instrument is designed to require minimum maintenance However to preserve the reliability of the instrument you must regularly perform basic preventive maintenance procedures This chapter explains routine cleaning procedures that will keep your instrument in good condition AA All biological specimens and materials coming into contact with them are considered biohazardous Handle as if capable of transmitting infection Dispose of waste using proper precautions and in accordance with local regulations Never pipette by mouth Wear suitable protective clothing eyewear and gloves Mi Tip A 5 solution of sodium hypochlorite can be substituted for undiluted bleach in the following cleaning procedures However higher concentrations of sodium hypochlorite and use of other cleaning agents might damage the in
96. on Trigon and Octagon Maps 6 Color Blue Octagon Default Configuration Map o oooooo o 2 Color Violet Trigon Default Configuration Map 5 6 Color Violet Octagon Default Configuration Map 4 2 Color UV Trigon Default Configuration Map oooooooooo o 3 Color Red Trigon Default Configuration Map o ooooomoo o 4 Color Red Octagon Default Configuration Map 2005 Appendix G Instrument Configuration Application Installing ICA Updating Your Default Configuration 2 cece eee eee eee Restoring Your Default Configuration 0 e eee eee eee ee Appendix H Setting Laser Delay Laser Delay Optimizing the Laser Delay Setting o oooooomomomomom o r Index X BD LSR II User s Guide 163 164 165 169 175 176 177 178 180 181 183 185 187 189 190 191 192 193 194 195 197 198 199 202 203 204 205 207 About This Guide The BD LSR II User s Guide describes the procedures necessary to operate and maintain your BD LSR II flow cytometer Because many instrument functions are controlled by BD FACSDiva software this guide also contains information about software features required for basic instrument setup and operation For an annotated list of additional documentation for your BD LSR II flow cytometer see BD LSR II Documentation on page xiii xi Conventions The following
97. oner to convert the local power source to meet the BD LSR II power requirements 120 V 10 50 60 Hz Contact your local BD office for further information General Safety A A The instrument handles are for BD Biosciences authorized personnel only Do not access them or attempt to lift the instrument with them or you could injure yourself To avoid burns do not touch the fan guards on the back of the instrument The fan guards could be hot during and after instrument operation Safety and Limitations xix Biological Safety XX AA All biological specimens and materials coming into contact with them are considered biohazardous Avoid exposure to biohazardous material by following these guidelines Handle all biological specimens and materials as if capable of transmitting infection Dispose of waste using proper precautions and in accordance with local regulations Never pipette by mouth Wear suitable protective clothing eyewear and gloves Expose waste container contents to bleach 10 of total volume for 30 minutes before disposal Dispose of waste in accordance with local regulations Use proper precaution and wear suitable protective clothing eyewear and gloves Prevent waste overflow by emptying the waste container frequently or whenever the waste management system alarms For information on laboratory safety refer to the following guidelines NCCLS documents can be ordered online at www nccls org P
98. ot Piot Title Content IV Tube IV Populations a Specimen m Title Font sansSerit Dad Size 12 Color o T taic 7 Bold 7 On the MyDataAnalysis global worksheet fluorescence plots e Draw a gate around the FITC positive population Name the population FITC positive e Draw a gate around the PE positive population Name the population PE positive e Draw a gate around the PerCP positive population Name the population PerCP positive e Draw a gate around the APC positive population Name the population APC positive 76 BD LSR II User s Guide 8 Format the Statistics view e Right click on the Statistics view and select Edit Statistics View e Under the Header tab specify displaying Specimen Name and Tube Name only e Under the Populations tab select all of the populations except All Events e Under the Statistics tab specify displaying the mean for each of the fluorescence parameters e Click OK Edit Statistics View Header Populations Statistics Parameters o E SD Mode E O O E O m m m m O O E E m mK IK Ikik im im Sort by Parameter C Sort by Formula 9 Print the analysis Your global worksheet analysis objects should now resemble those in Figure 3 15 on page 78 Chapter 3 Running Samples 77 Figure 3 15 Lymphocyte analysis Beads_001 All Events E Singlets 50
99. our workspace before recording data 1 Using the browser toolbar create a new Specimen in MyExperiment and rename it FourColorBeads 2 Create two Tubes for the FourColorBeads Specimen Rename the Tubes Beads_001 and Beads_002 3 Expand the Global Worksheets item to access your default global worksheet Rename the worksheet MyData 70 BD LSR II User s Guide Select Experiment gt Experiment Layout to display the Experiment Layout dialog This dialog is used to define parameter labels and to specify the number of events to record for each Tube e Parameter labels appear on the plot axes and in all statistics views For these bead samples you do not need to define additional parameter labels When you record immunophenotyping data use the Labels tab of the Experiment Layout dialog to provide reagent antibody labels e Click the Acquisition tab Specify 10 000 events to acquire for both Tubes Experiment Layout Labels Keywords 1 Events to Record 10 000 Y E MyExperiment EA FourColorBeads 10000 If Beads_001 10000 L U Beads_002 e Click OK Click the Template tool af on the Worksheet toolbar to switch to the normal worksheet view On the MyData worksheet create some plots for previewing data e FSC vs SSC e FITC vs PE e FITC vs PerCP e FITC vs APC Chapter 3 Running Samples 71 Recording Data In this section you preview and record data for multiple samples
100. parameter detector arrays and channel information for the selected configuration Figure G 1 on page 200 Click Upgrade to update your BD FACSDiva software default instrument configuration In the main text area a log of actions performed on the BD FACSDiva software instrument configuration database is appended to the previous configuration information display Figure G 2 on page 201 Appendix G Instrument Configuration Application 199 Figure G 1 ICA Select Configuration BD FACS Instrument Configuration Application 6 Violet 6 Violet 6 Violet Select Configuration LSRIl 4Blue 2Violet 2UY 2Red 6Blue OViolet OUV 3Red Currently selected configuration Parameter Octogon FSc ssc FITC PE PE Texas Red PE Cy5 PercP Cy5 5 PE Cy7 Alexa Fluor 488 CN gt Aa Indo 1 Blue Indo 1 Violet DAPI APC Alexa Fluor 700 APC Cy7 Currently selected instrument LSRII 6 Blue D Violet Channel NID OP DN ao 2 uv 200 BD LSR II User s Guide Figure G 2 I E Ez Fil Help ICA Upgrade Log BD FACS Instrument Configuration Application Upgrade Save Log Select Instrument select Configuration isan y 2 Violet Red Inserting Inserting Inserting Insertion Inserting Insertion Inserting Insertion Inserting Insertion Inserting Insertion Inserting Insertion Insertion Insertion Upgrade c 0 Violet iolet et 2 Violet 6 Violet 6
101. particles Make new QC samples and perform the quality control procedure again 130 BD LSR II User s Guide Instrument Troubleshooting continued Observation Possible Causes Recommended Solutions Poor QC results Air bubble or debris in flow Prime the fluidics system See cell Priming the Fluidics on page 51 Old or contaminated QC Make new QC samples and particles perform the quality control procedure again Sample not diluted in same Dilute the sample in the same fluid fluid as sheath fluid as you are using for sheath If you are running BD CaliBRITE beads dilute them in BD FACSFlow solution and use BD FACSFlow solution for sheath fluid Laser not warmed up Wait the recommended amount of time for the laser to warm up e 30 min for the 488 nm blue 60 min for the 325 nm UV e 30 min for the 355 nm UV e 15 min for the 405 nm violet e 20 min for the 633 nm red Laser not functioning Contact BD Biosciences Optical alignment problem Contact BD Biosciences Chapter 7 Troubleshooting 131 132 BD LSR II User s Guide Appendix A Technical Overview This appendix contains a technical overview of the following topics e Fluidics on page 134 e Optics on page 135 e Electronics on page 144 133 Fluidics The fluidics system in the BD LSR II flow cytometer is pressure driven a built in air pump provides a sheath pressure of 5 5 psi After passing through the sheath fil
102. re absorbance filters that do not have any specific reflective characteristics Also optical filters and beam splitters are rated at a specific angle of incidence When used in front of the fluorescence detectors they are perpendicular to the incident light and when used as a beam splitter they are placed at an angle relative to the light source Their optical properties are therefore designed for that angle of incidence Compensation Theory Fluorochromes emit light over a range of wavelengths recall Figure A 3 on page 137 Optical filters are used to limit the range of frequencies measured by a given detector However when two or more fluorochromes are used the overlap in wavelength ranges often makes it impossible for optical filters to isolate light from a given fluorochrome As a result light emitted from one fluorochrome appears in a detector intended for another Figure A 6 This is referred to as spillover Spillover can be corrected mathematically by using a method called compensation Appendix A Technical Overview 141 142 Figure A 6 Spillover from the FITC fluorochrome to the PE detector FITC PE DF53080 DF 575 26 normalized intensity 500 nm 550 nm 600 nm 650 nm 700 nm For example FITC emission appears primarily in the FITC detector but some of its fluorescence spills over into the PE detector The spillover is corrected or compensated for hence the term fluorescence compensation Figure A 6 shows that so
103. ris and is properly pressurized 134 BD LSR II User s Guide Figure A 1 Hydrodynamic focusing of the sample core through the flow cell low sample high sample pressure laser beam pressure laser beam 12 pL min 60 uL min sheath sheath fluid fluid sheath fluid sheath ml fluid sample Optics The optics system consists of lasers optical filters and detectors Lasers illuminate the cells or particles in the sample and optical filters direct the resulting light scatter and fluorescence signals to the appropriate detectors Light Scatter When a cell or particle passes through a focused laser beam laser light is scattered in all directions Figure A 2 on page 136 Light that scatters axial to the laser beam is called forward scatter FSC light that scatters perpendicular to the laser beam is called side scatter SSC FSC and SSC are related to certain physical properties of cells e FSC indicates relative differences in the size of the cells or particles e SC indicates relative differences in the internal complexity or granularity of the cells or particles Appendix A Technical Overview 135 Figure A 2 Forward scatter FSC and side scatter SSC side scatter light source forward scatter Fluorescence When cells or particles stained with fluorochrome conjugated antibodies or other dyes pass through a laser beam the dyes can absorb photons energy and be promoted to an
104. rotection of Laboratory Workers from Instrument Biohazards and Infectious Disease Transmitted by Blood Body Fluids and Tissue Approved Guideline Wayne PA National Committee for Clinical Laboratory Standards 1997 NCCLS document M29 A Procedures for the Handling and Processing of Blood Specimens Approved Guideline Wayne PA National Committee for Clinical Laboratory Standards 1990 NCCLS document H18 A BD LSR II User s Guide Symbols and Labels The following symbols warnings or precaution labels appear on the BD LSR II flow cytometer or the waste and fluid tanks Symbol Meaning Location s Dangerous voltage Rear instrument panel near power receptacle Laser radiation hazard Near all removable covers and any place where the laser beam can emerge from the instrument Caution Consult accompanying Near the instrument documents handles Label Meaning Location s Caution Rear instrument panel adjacent to exhaust fans CAUTION Hot Surface Hot surface ATTENTION Surface Chaude 334972 CAUTION e Rear instrument panel adjacent to power receptacle CAUTION Turn power off Turn power off before before service service ATTENTION i Mettre hors tension e Near internal avant toute intervention power shield 334974 Safety and Limitations xxi xxii Label Meaning Location s CAUTION Rear instrument CAUTION anel adjacent to High Voltage High voltage P
105. rs The first number indicates the center wavelength and the second refers to the width of the band of light that is passed For example a 500 50 BP filter transmits light that is centered at 500 nm and has a total bandwidth of 50 nm Therefore this filter transmits light between 475 and 525 nm Figure A 5 on page 140 Appendix A Technical Overview 139 Figure A 5 Bandpass filter bandpass 100 460 500 540 480 520 transmission 0 450 500 550 wavelength nm BP and DF filters have the same general function they transmit a relatively narrow band of light The principal difference between them is their construction DF filters have more cavities or layers of optical coatings resulting in a steeper transmission curve than the curve for a BP filter This steep slope means that a DF filter is better at blocking light outside the rated bandwidth of the filter 100 7 AS I BP 500 50 filter DF 500 50 filter S AF 500 50 filter 50 0 450 500 550 wavelength nm 140 BD LSR II User s Guide Dichroic Mirrors Dichroic filters that are used to direct different color light signals to different detectors are called dichroic mirrors or beam splitters Although dichroic mirrors have the properties of LP or SP optical filters you can not necessarily use any type of LP or SP filter as a beam splitter A beam splitter must have a surface coating that reflects certain wavelengths but many LP or SP filters a
106. sheath filter for trapped air bubbles 2 If bubbles are visible gently tap the filter body with your fingers to dislodge the bubbles and force them to the top 3 Pinch the vent line closed Chapter 2 Instrument Setup 49 Figure 2 5 Sheath filter cytometer fluid line roller clamp not visible vent line vent cap 4 Loosen the sheath filter vent cap to bleed off any air in the filter collect the excess fluid in a container 5 Replace the vent cap 6 Check the fluid lines for air bubbles 7 Open the roller clamp on the blue cytometer fluid line if necessary to bleed off any air in the lines 8 Close the roller clamp 50 BD LSR II User s Guide Priming the Fluidics Sometimes air bubbles and debris are stuck in the flow cell This is indicated by excessive noise in the forward scatter parameter In these cases it is necessary to prime the fluidics system 1 Remove the tube from the SIP 2 Press the PRIME fluid control button to force the fluid out of the flow cell and into the waste container Once drained the flow cell automatically fills with sheath fluid at a controlled rate to prevent bubble formation or entrapment The STNDBY button is yellow after completion 3 Install a 12 x 75 mm tube with 1 mL of DI water on the SIP and place the support arm under the tube 4 Repeat the priming procedure if needed Quality Control An instrument quality control QC procedure performed on a regular bas
107. strument Daily Cleaning and Shutdown Perform the following maintenance procedures every day e Daily Fluidics Cleaning on page 108 e Daily Shutdown on page 110 Daily Fluidics Cleaning Each time you shut down the instrument clean the sample injection tube and the area between the injection tube and the outer sleeve Figure 6 1 on page 109 This prevents the sample injection tube from becoming clogged and removes dyes that can remain in the tubing Vv Tip Follow this procedure immediately after running viscous samples or nucleic acid dyes such as Hoechst DAPI propidium iodide PI acridine orange AO or thiazole orange TO 108 BD LSR II User s Guide Figure 6 1 Sample injection port SIP Bal seal outer sleeve tube stop sample injection tube tube support arm 1 Set the fluid control to RUN 2 Install a tube containing 3 mL of a bleach solution on the SIP with the support arm to the side vacuum on and let it run for 1 minute Mi Tip For the bleach solution use BD FACS cleaning solution or a 1 10 dilution of bleach in DI water BD FACS cleaning solution is a bleach based cleaning agent designed for daily use in cytometer maintenance 3 Move the support arm under the tube vacuum off and allow the bleach solution to run for 5 minutes with the event rate set to HI 4 Repeat steps 2 and 3 with BD FACS rinsing solution BD FACS rinsing solution is a detergent based cleaning solution 5 Repeat
108. t Print Chapter 3 Running Samples 73 Figure 3 14 Recorded data showing singlet population FourColorBeads Beads 001 FourColorBeads Beads_001 50 100 150 200 250 258269 185946 FSC A x 1 000 FourColorBeads Beads_ 001 74 BD LSR II User s Guide Analyzing Data In this section you analyze the recorded Tubes by creating plots gates and population hierarchy and statistics views on a new global worksheet When complete your new global worksheet should resemble that in Figure 3 15 on page 78 1 Use the browser toolbar to create a new global worksheet Name it MyData Analysis 2 Create plots on the MyDataAnalysis worksheet e FSC vs SSC e FITC vs PE e FITC vs PerCP e FITC vs APC 3 Create Population Hierarchy and Statistics views e Select Populations gt Show Population Hierarchy Position the Population Hierarchy view below the plots e Select Populations gt Create Statistics View Position the Statistics view below the Population Hierarchy view 4 Draw a gate around the singlets on the FCS vs SSC plot Use the Population Hierarchy to rename the population Singlets 5 Select all plots except the FSC vs SSC plot Use the Inspector to specify showing only the Singlets population in the selected plots Chapter 3 Running Samples 75 6 Select all plots Click the Title tab in the Inspector and specify displaying only the Tube and Population names in the plot titles Inspector xj Plot Labets D
109. t of DNA fluorescence as singlet G M cells they accumulate in the same fluorescence area channels see Figure 4 1 above Therefore singlets and doublets must be distinguished to obtain cell cycle analysis accuracy Signal width vs area can be employed to accurately identify aggregate events DNA Setup In this chapter you use the BD DNA QC Particles kit to verify critical DNA analysis criteria and optimize your cytometer for DNA experiments The instructions given here assume that DAPI is being used as described in the section How to Use DAPI with DNA QC below If you are using PI for DNA QC see the section How to Use PI with DNA QC and substitute PI for DAPI in subsequent instructions of the chapter How to Use DAPI with DNA QC Before beginning this chapter do the following e Prepare biological standards for instrument quality control using the BD DNA QC Particles kit Substitute the PI solution in the BD DNA QC Particles kit with a 1 0 nanomolar nM DAPI solution prepared in 1 BSA e Prepare one tube each of chicken erythrocyte nuclei CEN and calf thymocyte nuclei CTN sample according to the kit instructions Substitute the DAPI solution for the PI solution The CEN sample is used to check instrument resolution CV and linearity The CTN sample is used to verify the system s ability to resolve singlets from aggregates Chapter 4 DNA Analysis 83 How to Use Pl with DNA QC You can use the PI that com
110. tations Laser Safety usais a as Laser Classification o oooooomoo o o Precautions for Safe Operation Electrical Safety ooooooooomomo mm General Safety aa cia a a bas Biological Safety o oooooomomomm Symbols and Labels o oooooo o o Limitations aa y eS Chapter 1 Introduction OVERVIEW A te eis ets wee eee E E CompoOnienits A Bie Na cea aie T IM ENEA Power Switch s 6 0 800 esa cha eds ee Ses Control Panel cc ee ised qa e eae dina Handles ss sea ak Ba sleet Oh Ab os AAAS bees Aas Ba awe ah Be Fluidi s aesaat 0h se Ge e Ga Ra eee ea ea Sample Flow Rate Control 0 0 cece cece ences Fluid Control 32 sts otc tik Beek BA ae a ROM ae eed Be Sample Injection Port 0 cece cece oo Sheath and Waste Containers 0 0c eee ee cee eee eee tenes DETECTORS 3 MAR Bie etek BR eee MAL ocr ABN a Bb ie ae tA ak ti BD LSR II Workstation osason ccc cece eee eee eens Chapter 2 Instrument Setup vi Starting the Flow Cytometer and Computer 00 0 cece eee Setting Up the Optical Filters and Mirrors 0 000 e cence eens Filter and Mirror Configurations 0 ccc cece eee eens Changing Optical Filters or Mirrors 02 0 cece eee eee ee eee Filter and Mirror Specifications 02 cece eee eee eee eens Preparing Sheath and Waste Containers 0 0 ce cece eee eens Preparing the Sheath Container 00 0 e ce
111. tatistics View e Under the Population tab deselect Events and Parent e Under the Statistics tab select the mean for Indo 1 Blue A Indo 1 Violet A and the ratio parameter e Click OK In the Acquisition Controls frame set the Events To Record to 1 000 000 evts and the Events To Display to 500 evts FE Acquisition Controls x CaF_001 0 evtis 00 00 00 de E Acquire E Record Restart Storage Gate m All Events v Stopping Gate m All Events v Events To Record 1000000 evt nd Events To Display 500 evt Only the specified number of Events to Display are shown during acquisition and recording After data recording is complete all recorded events will be displayed Optimizing for Calcium Flux 1 2 On the control panel press RUN and LO Install the sample not stimulated yet on the SIP Verify that the green Acquisition pointer is in front of the CaF_001 Tube in the Browser In the Acquisition Controls frame click Acquire Chapter 5 Calcium Flux 101 4 Adjust the FSC and SSC voltages to place the lymphocytes on scale in the FSC vs SSC dot plot 5 Adjust the FSC threshold to remove debris without cutting into the lymphocyte population 6 Draw a gate around the lymphocytes Create a Population Hierarchy view and rename the population Lymphocytes Figure 5 2 Figure 5 2 Lymphocyte gate CaF _001 Lymphocytes _ 1 000 SSC A Lymphocytes 50 100 160 200 260 FSC
112. ter sheath fluid is introduced into the lower chamber of the quartz flow cell The sample to be analyzed arrives in a separate pressurized stream When a sample tube is placed on the sample injection port SIP the sample is forced up and injected into the lower chamber of the flow cell by a slight overpressure relative to the sheath fluid The conical shape of the lower chamber creates a laminar sheath flow that carries the sample core upward through the center of the flow cell where the particles to be measured are intercepted by the laser beam Figure A 1 on page 135 This process is known as hydrodynamic focusing The objective in flow cytometric analysis is to have at most one cell or particle moving through a laser beam at a given time The difference in pressure between the sample stream and sheath fluid stream can be used to vary the diameter of the sample core Increasing the sample pressure increases the core diameter and therefore the flow rate Figure A 1 on page 135 e A higher flow rate is generally used for qualitative measurements such as immunophenotyping The data is less resolved but is acquired more quickly e A lower flow rate is generally used in applications where greater resolution and quantitative measurements are critical such as DNA analysis Proper operation of fluidic components is critical for particles to intercept the laser beam properly Always ensure that the fluidics system is free of air bubbles and deb
113. the ratio over time reflects the increase in intracellular Ca concentration Figure 5 1 Calcium flux data CaF_001 Lymphocytes lu 3 a z a Lu 3 250 x 1 000 BD LSR II User s Guide Calcium Flux Optimization Before beginning this section do the following e Start up the instrument and perform QC e Ensure that the appropriate filters are installed See Setting Up the Optical Filters and Mirrors on page 41 e Review the following section Using the Time Parameter M Tip For calcium flux experiments use a sheath fluid that does not contain preservatives For example use 1X phosphate buffered saline PBS Using the Time Parameter The Time parameter shows how events change over time In calcium flux experiments the Time parameter displays the rate at which the cells in the sample respond to a stimulus The Time parameter is a fixed scale and cannot be altered The values for the Time parameter are in 10 ms increments thus for a given event a Time parameter of 123 represents 1 230 ms Because a plot spans 2 6 262 143 tickmarks will span 2 6 2 621 430 ms or 44 minutes Therefore it takes an event approximately 44 minutes to travel from one end of a plot to the other Vv Tip To allow enough time for Ca flux response and resolution enter a large value for the Events to record before recording events You cannot enter a specific time in which to record events or assign a time resolution
114. tics cover and laser shielding in any way A A only BD Biosciences service personnel should perform service procedures on the laser equipment and the BD LSR II instrument Precautions for Safe Operation To reduce the possibility of hazardous exposure to radiant laser energy do not remove caution labels from the instrument Caution labels are located on all removable covers or any place where the laser beam can emerge from the instrument A ZA Use of controls or adjustments or performance of procedures other than those specified in the BD LSR II User s Guide can result in hazardous radiation exposure xviii BD LSR II User s Guide Electrical Safety A A Lethal electrical hazards can be present in all lasers particularly in laser power supplies Every portion of the electrical system including the printed circuit boards should be considered to be at a dangerous voltage level Avoid potential shock by following these guidelines Connect the equipment only to an approved power source Do not use extension cords Have an electrician immediately replace any damaged cords plugs or cables Refer to the BD LSR II Facilities Requirement Guide for specific information Do not remove the grounding prong from the power plug Have a qualified electrician replace any ungrounded receptacles with properly grounded receptacles in accordance with the local electrical code For installation outside the US use a power transformer or conditi
115. timizing the red laser set its delay to 60 psec 6 While observing the positive events on the histogram plot adjust the Laser Delay setting in 1 psec increments within a range of 10 psec of the initial setting Choose the setting that moves the events farthest to the right highest fluorescence intensity 7 Draw an Interval gate on the histogram plot for the positive events Create a Statistics view to display the mean fluorescence intensity of the gated population 8 While observing the mean fluorescence intensity for the gated population adjust the Laser Delay setting in 0 1 psec increments within a range of 2 0 psec of the setting obtained in step 6 You should also stay within a range of 10 psec of the initial setting step 5 Preserve the setting that maximizes the fluorescence intensity 9 Reset the Window Extension value to 10 psec 206 BD LSR II User s Guide Index Numerics 325 nm or 355 nm laser 33 405 nm laser 33 488 nm laser 33 43 633 nm laser 33 A Acquisition Templates creating 70 previewing data 70 79 acridine orange AO 108 adjusting gate boundaries 64 threshold 63 voltages 63 air in filter removing 49 50 alarm waste container 31 ALPHA filters AFs 34 analysis batch 54 data 70 DNA 82 immunophenotyping 75 reusing 79 saving 79 antibodies 136 applications calcium flux 96 DNA 82 Apply to All Compensation Tubes 64 Bal seal replacing 118 bandpass BP filters 34 139 diameter 4
116. trol on page 28 Figure 1 2 Control panel SAMPLE FINE ADJ Ly e e sample flow rate control buttons fluid control buttons Handles A The instrument handles see Figure 1 1 on page 26 are for BD Biosciences authorized personnel only Do not access them or attempt to lift the instrument with them or you could injure yourself Chapter 1 Introduction 27 Fluidics 28 The purpose of the fluidics system is to carry the sample out of the sample tube and into the sensing region of the flow cell Cells are carried in the sample core stream in single file and measured individually Sample Flow Rate Control Three flow rate control buttons LO MED and HI set the sample flow rate through the flow cell The SAMPLE FINE ADJ knob allows you to adjust the rate to intermediate levels When the SAMPLE FINE ADJ knob is at its midpoint the sample flow rates at the LO MED and HI settings are approximately 12 35 and 60 pL min of sample respectively The knob turns five full revolutions in either direction from its midpoint providing sample flow rates from 0 5 2X the midpoint value For example if the LO button is pressed the knob will give flow rates from approximately 6 24 pL min Fluid Control Three fluid control buttons RUN STNDBY and PRIME set the instrument mode Figure 1 2 on page 27 RUN pressurizes the sample tube to transport the sample through the sample injection tube and into th
117. ube tube support arm Chapter 1 Introduction 29 30 e Sample injection tube stainless steel tube that carries sample from the sample tube to the flow cell This tube is covered with an outer sleeve that serves as part of the droplet containment system e Tube support arm arm that supports the sample tube and activates the droplet containment system vacuum The vacuum is on when the arm is positioned to the side and off when the arm is centered Droplet Containment System The droplet containment system prevents sheath fluid from dripping from the SIP and provides biohazard protection When no sample tube is installed on the SIP sheath fluid backflushes through the sample injection tube This backflush helps prevent carryover of cells between samples The droplet containment system vacuum is activated when the sample tube is removed and the tube support arm is moved to the side Sheath fluid is aspirated as it backflushes the sample injection tube If a sample tube is left on the SIP with the tube support arm to the side vacuum on sample will be aspirated into the waste container BD LSR II User s Guide Sheath and Waste Containers The sheath and waste containers are outside the instrument and can be positioned on the floor Sheath Container The sheath container has a capacity of 8 L Sheath fluid is filtered through an in line interchangeable filter that prevents small particles from entering the sheath fluid lin
118. uration defines which fluorochrome or scatter parameter is measured at each instrument detector BD FACSDiva software provides a default instrument configuration software construct based on your BD LSR II Use the BD FACSDiva Instrument Configuration dialog box to modify delete and create new instrument configurations See the Instrument and Acquisition Controls chapter of the BD FACSDiva Software Reference Manual for details Reconstructing the BD FACSDiva Default Instrument Configuration With the BD FACSDiva Instrument Configuration dialog box it is possible to accidentally delete your default instrument configuration You may want to have a series of acquisitions based on identical or compatible having an identical parameter subset instrument configurations Therefore if you accidentally delete your default instrument configuration you are likely to want to reconstruct it to exactly match the original any change in parameter Chapter 2 Instrument Setup 43 44 labels can nullify the configuration compatibility The BD FACS Instrument Configuration Application ICA can restore your default configuration Instructions for using ICA software are found in Appendix F BD LSR II Configuration Upgrade If you upgrade your BD LSR II cytometer with additional lasers or detector arrays you will need to run the ICA software to update the BD FACSDiva database for your instrument Instructions for installing and using the ICA softwar
119. yed in Figure 3 2 all checkboxes deselected Figure 3 2 User Preferences dialog ff User Preferences e Under the Templates tab verify that the Default global worksheet checkbox is enabled See the BD FACSDiva Software Reference Manual for more information about the instrument configuration and user preferences 58 BD LSR II User s Guide Creating an Experiment In this section you create an Experiment in a new folder specify the parameters for Instrument Setup and add compensation Tubes 1 Use the buttons in the Workspace to display the frames listed below Browser Instrument Inspector a aa Worksheet TAS Acquisition Controls 2 Use the New Folder button in the Browser toolbar Figure 3 3 to add a new folder to the Browser Rename the folder MyFolder Figure 3 3 Browser buttons New Folder New Specimen New Instrument Settings _ New Plate ES New Experiment NewTube New Global Worksheet Select Plate Type M Tip To place an Experiment inside a folder select the folder before creating the Experiment 3 Select MyFolder by clicking its folder icon in the Browser and then use the New Experiment button in the Browser toolbar to create a new Experiment Rename the Experiment MyExperiment Chapter 3 Running Samples 59 60 4 With the Experiment selected verify in the Inspector frame that the Use global instrument settings checkbox is enabled Figure 3 4 Use global instrument sett

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