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FACSCalibur, and FACSVantage-SE SOPJanuary 6, 2011Flow Cytometer FACSCalibur and FACSVantage flowsorterStandard Operating ProceduresFACS FacilityBuilding 977 – 106January 10, 2011Please Note: These procedures will be updated as needed; with the current versionalways posted in the FACS facility, on the instrumentation. Users need to be ensuringthat they always use the current, posted version.Prepared By:Damir Sudar, Life Sciences DivisionMichelle Scott, Life Sciences Division1
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 2011Table of Contents1. Research Description1.1. Work Overview1.2. Flow cytometer and Sorter (FACS) Analysis Summary1.3. How to Use the BSL2 FACS Facility2. Responsibilities3. Determination of Hazards and Controls3.1. HazardsBiohazardous MaterialsLasersPressure3.2. ControlsAdministrativeTrainingWork PracticesEngineeringBiosafety CabinetFAC HEPA Exhaust SystemPersonal Protective Equipment4. FACSCalibur Operating Procedure4.1. System Check4.2. Running a Sample5. FACVantage Operating Procedures5.1. System Check5.2. Running a Sample6. FACSVantage Preventive Maintenance Procedures7. AppendixA: FACS Users Log (Excel Spread Sheet)B: FSE Tools/Supplies Required2
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 20111.0Research DescriptionSee the appropriate section of users BUA1.1Work OverviewCell flow instruments and the technical support and expertise will be available to theLBNL and external research community. These instruments include a Becton-Dickinson(BD) FACSVantage-SE high speed cell sorter, and a BD FACSCalibur bench top flowcytometer. The FACSVantage-SE will sort or isolate subpopulations of cells from avariety of sources including cells with specific reporters, stem cells, or primary tissues(blood or other human or mouse tissues or tumors).All researchers wishing to have samples processed on these instruments will transporttheir FACS-ready samples to the FACS facility in 977-106 following EH&S guidelines forcontainment and safety. The samples will not be stored or incubated on site, butimmediately processed on the cytometer/sorter by a well-trained manager, MichelleScott (977-118 ext4281).To maintain a record for EH&S, we require that each user fill out a form delineating whatbiological material is being analyzed and to disclose any potential biohazards. Thisrequest form (Appendix A) has been developed in collaboration with EH&S as a recordof material exposure, and will be kept on file. Requests that entail unknown biohazardsare referred to EH&S for review. No radioactive material is allowed in the facility. OnceFACS users have gone through extensive, certified FACS training, they may be able toprocess their own samples.1.2Flow cytometer and Sorter (FACS) Analysis SummaryThe Becton-Dickinson (BD) FACSVantage SE flow cytometer sorter can sort/isolate or measuresubpopulations of cells from a variety of sources including cells with specific reporters, stemcells, or primary tissues (blood or other human or mouse tissues or tumors). Only RG 1 and 2materials will be processed.The FACS systems are located in a BSL2 lab in 977-106. Users must ensure proper protectiveclothing is worn at all times. While in 977-106, closed toe shoes and safety glasses arerequired at all times. When working with the sorters, or handling or sorting all biologicalspecimens: closed toe shoes, safety glasses, lab coat, and appropriate gloves are required atall times.All researchers wishing to use the FACS facility must ensure that they have taken all requiredEH&S training and that the FACS Standard Operating Procedure document, method of sampletransport to the FACs facility, and trained users have been documented in their PIs BUA, beforescheduling an experiment on the Oracle calendar. When scheduling, it is required that usersgive details of the samples that will be sorted under the “details” tab, listing any potentialhazards.All researchers wishing to have samples processed on these instruments will transport theirFACS-ready samples to the FACS facility in 977-106 following EH&S guidelines for containmentand safety as outlined in their respective PIs BUAs. The samples will not be stored or incubated3
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 2011on site, but immediately processed on the cytometer/sorter. Researchers, who have not yetbeen trained, will schedule processing with the FACS manager, Michelle Scott. Researchers,who have passed extensive FACS training, will be certified by the FACS manager in theCertified User Log. They will then have their instrument login activated so that they can processtheir own samples.Users are required to fully document and sign-in in the Certified User Log before eachexperiment. If this requirement is not met, users will lose FACS privileges. A computer log willautomatically be generated with each FACS run and checked against the Certified User Log.To maintain a record for EH&S, before each run each user is required to sign in to the user logand disclose any potential biohazards. This log has been developed in collaboration with EH&Sas a record of material exposure, and will be kept on file. If the potential biohazards of a sampleare not well understood, the user is to confer with EH&S prior to sample processing. Noradioactive material is allowed in the facility.Only trained and authorized users as documented in the Certified User Log-Book are allowed tooperate the FACS systems.The manager and any trained and authorized users will at all times follow the FACS StandardOperating Procedures (SOP) for 1) system operation and 2) running a sample for each of theinstruments in the facility. The SOPs are maintained and available in the FACS facility with thecurrent master copies posted next to each instrument. Information in the SOPs may change andall users are expected to familiarize themselves with any changes.1.3How to Use the BSL2 FACS Facility(1) The appropriate red "warning sections" on protective measures are at the beginning of eachSOP. Users are required to follow these and all BSL2 safety procedures when they are inthe FACS facility.(2) A calendaring step has been added to the summary which requires calendaring in ORACLE.In the "details" section of the entry, the user describes the sample in enough detail thatpotential biohazards are described.(3) The Certified User Log has been divided into an excel file with 6 tabs. The first 3 tabs (1 perinstrument) is the running list where the user signs in for a run (columns were added for theuser to describe the sample and potential biohazards, and to put their signature). The last 3tabs (1 per instrument) is completed by Sarah and is the list of Authorized/trained users(columns were added for the user and trainer to put their signatures).(4) Prior to Training:(a) Each user checks that they are have completed all EH&S training courses to handleRG2 level samples (BSL2 safety procedures are always followed in the facility).(b) The user puts a copy of these Standard Operating Procedures in their PIs BUAadding a statement of how they will safely transport their samples from the PIslaboratory to the FACS facility.(c) If they are trained, they add their name as a "FACS VANTAGE ( or FACS Calibur) user"on their PIs BUA (next time BUA is renewed - until then it is listed in the Certified Log in106).4
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 2011(5) When a user wants to run:(a) They calendar their run (describing the potential hazards in "details")(b) If they will need an Operator for their run, they notify Sarah of their run by email (at leasta week in advance).(c) The user brings his samples and all needed materials to the facility (gloves, tubes,pipettes etc); the user signs in fully on the Log sheet.(d) The user verifies that the instrument was left in the correct shutdown state; they maywant to run a cleaning cycle before they start, just to be sure it is ready.(e) The instrument is operated, shut down and cleaned, and the samples are processedaccording to the current, authorized SOP (copy on instrument).(f) The user signs out with the time of completion on the Log.(g) The user discards all biohazardous waste in the appropriate receptacle (and logs thecontents on the sheet affixed to the can), discards all other waste and thoroughly cleansup the bench area (using a solution of 10% household bleach and then 70% ethanol).(h) Wash hands at the sink before gathering up items and leaving the facility.(i) Please let Sarah know if any problems were encountered.Remember for users to be allowed access to the facility, it is critical that they practice stringentBSL2 procedures, follow the SOPs, and thoroughly clean up after themselves.2.0Responsibilities Michelle Scott is the FACS facility manager and is responsible for the operationand maintenance of the FACS systems, while Damir Sudar provides oversight. FACS users will be responsible for keeping all required EH&S training updatedand using only the authorized, current FACS SOPs posted in the FACS facility.They are also responsible for adding the following to their PIs BUA: (1) the FACSFlow Cytometer Sorter (FACS) Calibur and Scanner Standard OperatingProcedures document, (2) documentation of sample preparation and transport tothe FACS facility, (3) FACS users names and training level.3.0Determination of Hazards and Controls3.1HazardsThe main hazards associated with use of this equipment involves: biological materials,lasers and pressure.Biological Materials:The projects carried out in the FACS facility (977-106) involve the use of both RiskGroup 1 (RG1) and RG2 biological agents-materials; and will all be carried out usingstandard Biosafety Level 2 (BL2) work practice controls. Personnel handling humantissues, human blood, and human cells will assume that bloodborne pathogens could bepresent and will handle these materials as if they are potentially infectious using“universal precautions.” Personnel handling primary human tissues, cells and viruses willbe very limited and are noted in the body of their PIs BUA. The transfer of any biological5
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 2011materials to the FACS facility will occur using BSL2 procedures, including sealed, leakproof, secondary containment, gloves, eye protection and lab coat. Clean gloves will beused to open the doors between the labs.Lasers: The instruments contain embedded lasers to which the class 1 laser protocolapplies as approved by LBNL‟s Laser Safety Officer (LSO). Under the class 1 embeddedlaser protocol, only approved and licensed vendor technicians are allowed open beamalignment and repair activities. LBNL personnel will NOT perform these activities at anytime but will provide oversight when vendor service personnel performs these activities.The FACS facility manager will be present at such time.Pressure: The FACS Vantage utilizes a 100 um nozzle to generate pressures up to 2021 psig. Injury may occur from the rupture of high pressure lines. Additional, materialsbeing sorted can easily be aerosolized and become a inhalation hazard.3.2 ControlsAs mentioned previously, aspects of this project are conducted in a manual fashion inthe laboratories listed, and are therefore authorized by the individual PI‟s BUAs.3.2.1Administrative ControlsTraining: Training of all personnel using the FACS will be documented and coveredunder the BUA issued to their PI. Users are responsible for ensuring that theirEH&S training and FACS training is complete and documented in their PIsBUA. Certified training provided by FACS staff on the FACS instrument ofchoice will be required before login rights are activated and independentaccess is allowed.Work Practices: Standard BL2 practices will be used. All human or murine cell lines willbe handled under BL2 containment. Hand washing will be required. Use of personal protective equipment (gloves, lab coats and safetyglasses) is required. Surface disinfection (10% solution of household bleach), will beperformed before and after each run. Agents being removed from these instruments will be either:(1) Handled as biohazardous waste (biohazard waste container andlog in 977-109),(2) Decontaminated by flushing/spraying with a 10% solution ofhousehold bleach or(3) Contained in closed, leak-proof containers for storage,centrifugation, or other subsequent processing. The FACs instrumentation/tubing is disinfected by flushing with a 10%solution of household bleach. Liquid biological waste is mixed with6
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 2011 3.2.2household bleach to a 10% concentration and held for at least 15 minprior to drain disposal.All of the solid biological waste (both RG 1 & 2) is handled asbiohazardous waste and placed into red biohazard bags, which are thentaken off site and treated by LBNL‟s licensed medical waste contractor.Bleach and quaternary ammonium compounds are effective surfacedisinfectants against HIV-1 and all cell lines used in this project.After work is completed, all work surfaces are sprayed with a 10%solution of household bleach and then 70% ethanol.Engineer Controls:Biosafety Cabinets:BL2 containment within a biosafety cabinet or the ACCS unit in 977-116 will be usedfor all work involving the growth, treatment, transfection and harvesting of human ormurine cell lines.FACS High Efficiency Particulate Air (HEPA) Filtered Exhaust:The FACS utilizes a stand-alone HEPA filtering exhaust unit to capture any aerosolsgenerated by the FACS machine. To ensure the unit is properly operating, thesettings on the unit must be checked prior to sampling. This unit is also included inthe Preventative Maintenance (PM) program and must be HEPA tested and certifiedfor use before it can be placed back into service.3.2.3Personal Protective Equipment:All personnel working in 977-106 will don the following personal protectiveequipment: Closed toe shoes and safety glasses are required in 977-106. When working withthe sorters, or handling or sorting all biological specimens: closed toe shoes, safetyglasses, lab coat, and appropriate gloves are required at all times. Double gloves will be worn at all times when handling unfixed primary cells,lentiviral preparations, transfected cells, or the combined transfectionreagent.7
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 20114.0FACSCalibur Operating Procedures4.1System Check ProtocolOnly trained and authorized users, asdocumented in the Certified User Log, areallowed to operate the FACS Calibur .Researchers are responsible for ensuringthat all EH&S training and FACS BUApaperwork in their PIs laboratory is inorder, before scheduling an experiment.Users are required to calendar and sign inbefore each experiment.If these requirements are not met, userswill lose FACS privileges.The FACS systems are located in a BSL2lab. Users must ensure proper protectiveclothing (e.g., closed toe shoes lab coat,disposable gloves, and safety glasses) isworn at all times.Users are responsible for transportingtheir samples to the FACS followingEH&S guidelines for containment andsafety. They must document this method,their FACS training and FACS SOPs intheir PIs BUA.Secondary containment in a sealedcontainer (that won‟t leak if dropped) iscritical.All FACS waste will be consideredbiohazardous waste and must be properlyplaced in the correct waste stream.8
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 2011FACSCalibur SOPTurn on/off and system check protocolTURN ON: FACS Flow unit.Main power switch on cytometer.Macintosh workstation computer.To Log-on to the computer:Mac: Username: your login Password: your password The FACS CellQuest Pro software can be launched at this point.START UP DECONTAMINATIONAll work surfaces should be decontamined with an appropriate disinfectant prior toinitiating any work and after the machines have been place in operation.The Stream: Take the water tube off of the tube holder.Push button STAND-BY.Push button PRIME (wait till returns to STANDBY), repeat.QC: Prepare tubes of CaliBrite beads according to kit insert.Push button RUN and LOW.Guide the CaliBrite beads tube onto the SIP (Sample Insertion Port) until a firmseal is made. Swing tube support arm under tube.Follow FACScomp instructions on monitor.Use the BD FACS Comp software to calibrate and QC the system.CellQuest Pro SOFTWARE: If necessary, re-launch the software.FACSCalibur SHUT-DOWN: Remove sample tube.Install a tube of 10% bleach.Set the system to RUN, set flow to HIGH, run for 3 minutes.Put the system to STAND-BY and remove the bleach.Install a tube of dH2O.Set the system to RUN, set flow to HIGH, run for 3 minutes.Put the system in STAND-BY (leave tube of H2O on SIP)Turn of Computer Power.Turn off the main power switch.Turn off the FACS Flow unit.9
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 2011Shut Down Decontamination:All work surfaces must be decontaminated with appropriate disinfectant at the end ofeach day‟s run.Mac: Quit the CellQuest Pro software. Shut down the computer.10
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 20114.2Running a SampleOnly trained and authorized users asdocumented in the Certified User Log areallowed to operate the FACSCaliburResearchers are responsible for ensuringthat all EH&S training and FACS BUApaperwork in their PIs laboratory is inorder, before scheduling an experiment.Users are required to calendar and sign inbefore each experiment.If these requirements are not met, userswill lose FACS privileges.The FACS systems are located in a BSL2lab. Users must ensure proper protectiveclothing (e.g., closed toe shoes lab coat,disposable gloves, and safety glasses) isworn at all times.Users are responsible for transportingtheir samples to the FACS followingEH&S guidelines for containment andsafety. They must document this method,their FACS training and FACS SOPs intheir PIs BUA.Secondary containment in a sealedcontainer (that won‟t leak if dropped) iscritical.All FACS waste will be consideredbiohazardous waste and must be properlyplaced in the correct waste stream.11
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 2011FACSCalibur SOPRunning a SampleAll samples to be run on the BD FACSCalibur must be in a single cell suspension. Runcells through a 40um filter prior to arriving at the sorter to achieve this if they areclumping together.Only 5ml polystyrene round-bottom Falcon tubes can be inserted on the up-take port.(Cat # 352054). Uncap sample tube and place cap on an alcohol pad.Make sure the system is set to Stand-By.Carefully slide sample tube onto the SIP (Sample Insertion Port) and lift the tubeup over the pressure seal.Move the bottom arm beneath the tube. If necessary, adjust the height of thebase by turning it.Set the system to Run set flow to LOW or MED or HIGH.Once events are seen on the plots in the worksheet, you can make adjustmentsto voltages, compensation and threshold values.When all settings are as you want them, click „Record‟.When recording has finished, remove tube and set to STANDBY.Gates can be set on the populations of interest in the software.At the end of the run, turn the dial to Stand-By, cap & remove all collection tubes.Proceed with the shut-down procedure.12
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 20115.0FACVantage Operating Procedures5.1System Check ProtocolOnly trained and authorized users, asdocumented in the Certified User Log, areallowed to operate the FACS Vantage.Researchers are responsible for ensuringthat all EH&S training and FACS BUApaperwork in their PIs laboratory is inorder, before scheduling an experiment.Users are required to calendar and sign inbefore each experiment.If these requirements are not met, userswill lose FACS privileges.The FACS systems are located in a BSL2lab. Users must ensure proper protectiveclothing (e.g., closed toe shoes lab coat,disposable gloves, and safety glasses) isworn at all times.Users are responsible for transportingtheir samples to the FACS followingEH&S guidelines for containment andsafety. They must document this method,their FACS training and FACS SOPs intheir PIs BUA.Secondary containment in a sealedcontainer (that won‟t leak if dropped) iscritical.All FACS waste will be consideredbiohazardous waste and must be properlyplaced in the correct waste stream.13
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 2011FACSVANTAGE SOPTurn on/off and system check protocolStarting Up the InstrumentFollow these instructions to start up the instrument for operation in digital mode.1. Open the vacuum source and air supply.2. Verify that the sheath container is full and the waste container is empty. Ifneeded, empty the waste.3. Turn on the cytometer main power switch.(Lasers turn on automatically)4. Switch on the digital control switch, if necessary.5. The switch must be On to operate in digital mode.6. Turn on the computer main power switch and start up the BD FACSDivaworkstation.7. After logging on to Windows, launch BD FACSDiva software bydoubleclicking the shortcut on the desktop. Verify that the instrument isconnected by checking the Instrument window in the software. Themessage Instrument Connected appears after the cytometer connects tothe workstation (this can take several minutes).To Log-on to the computer:PC:Ctrl / Alt / DeleteUsername: AdministratorPassword: BDISLog on to: CC-S171-FACS VANTAGE (This computer)The FACS DIVA software can be launched at this point.Username: AdministratorPassword: (no password needed)8. If the Instrument Disconnected message remains, refer to thetroubleshooting suggestions in the software manual. All work surfaces should be decontamined with an appropriate disinfectant prior to initiating any work and after the machines have been place in operation.(OPTIONAL) Aerosol Containment Unit with the power button on the front. Themain power switch at the back on the bottom of the unit should always be on.The magnahelic gauge needle should be around 2. If not, the system powerlevel should be changed to bring the indicator to the right level. Verify thatthe magnahelic gauge indicates between the two black marks.Check that the lights on the front of the unit indicate that thefilter is still good and that it does not need to be replaced.The Digital switch should be ON.Digital Electronics ModuleThe digital electronics, oscilloscope, BD FACS Accudrop monitor, and digital14
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 2011control switch are housed within the digital electronics module (Figure 1-2). Theelectronics are adjusted by your field service engineer during installation and donot require user maintenance.Digital OperationDuring operation in digital mode, analog settings for gain, threshold,compensation, and the event rate are displayed on the analog oscilloscope.Because threshold and gain are controlled differently depending on the electronicmode, these settings can differ from those displayed by the digital electronics. For anaccurate event count, monitor the Acquisition Dashboard in BD FACSDivasoftware, rather than the analog oscilloscope.NOTICE In digital mode, most controls on the instrument control panel areinactive (see Figure 1-4). Equivalent controls can now be found in BD FACSDivasoftware.15
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 2011The STREAM: Fill Sheath Tank with PBS, empty Waste Tank.Disconnect pressure line and feed line from ETOH tank and connect them toSheath Tank.Turn on the pressure switch. (Behind the tube holder)With the 100 um nozzle, the sheath pressure should be 10-11 PSI.The Sample Differential should be 1.2-1.5 PSI for running samples.Turn the dial to FILL then STAND-BY.Allow stream to run for at least 15 minutes to flush ETOH out of system.Set the dial to RUN. It will drip from the up-take tubing.To flush the system: (Only if necessary) Place a cup or plastic holder beneath the syringe. (Next to tube holder) Turn the dial to STAND-BY Hold the syringe plunger in. Rotate the stopcock 180 from pointing to the instrument to pointing to yourself. Sheath will pour out from beneath the stopcock. This flushes the system ofcleaning solutions such as ethanol or bleach and also bubbles. Do this for 30 seconds. The return the stopcock to original position. (pointingtowards instrument.) To flush the nozzle, put the dial to FILL, press the NOZZLE FLUSH button. For more forceful flushing of the nozzle, put the dial to STAND-BY.16
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 2011 Rotate the stopcock 90 to point down.The syringe will fill. Keep thumb on the plunger. When full, turn the dial to FILL.Push the plunger in.Repeat several times, by turning the dial to STAND-BY to fill the syringe and thento FILL and pushing on the plunger.With the plunger in the pushed in position, rotate the stopcock back to face theinstrument.ALIGNMENT: (Usually only when necessary) Turn dial to STAND-BY.Guide a tube of rainbow beads onto the SIP (Sample Injection Port), keeping adrop of sheath at the end of the tubing so that no air comes between the tubingand the bead solution.Dial to RUN.To speed the beads through, temporarily increase Differential Pressure to 2.0.Adjust the Sample Differential to get the beads going through at desired rate of1000 Threshold Events/Second.17
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 2011 For 488 laser alignment, you are moving the sort block. Use the X dial, the largedial in the front near the Y dial, the large dial inside the side door, at the top nearthe left, and the Fluorescence Channel wheel.Laser alignment only requires access to the externally accessible knobs on theturning towers. Noopen beam alignment is allowed byLBNL personnel. If proper alignment cannot be achieved using thealignment knobs, call Michelle Scott ext. 4281 .18
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FACSCalibur, and FACSVantage-SE SOPJanuary 6, 2011 (Only done by Michelle Scott) Align the second and third lasers, one at a timeby adjusting the specific alignment knobs for each laser. Be sure to change theoscilloscope parameters to be reflecting the correct detection channel. Again,adjust alignment until the events are showing as high & tight as possibleLaser Delay: Set by Michelle ScottDIVA SOFTWARE: If necessary, re-launch the DIVA software. Make sure that Digital switch is ON. Change oscilloscope to dot plot.Note: When the DIVA software is not connected, the stream window appears dark.When it is connected, you can see the illuminated stream window. Turn dial to RUN for the rainbow beads.Have the oscilloscope set to FSC v SSC.27
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 2011 The events will show near the bottom of the screen until you click on „ACQUIRE‟.Then they will pop up to where you can see them.Note that on the computer screen in the DIVA software, the signal is in a „normalposition, however on the oscilloscope FSC and/or the SSC will be very high.Decrease SSC gain from 400 to 100. Decrease FSC gain from 800 to 100 or200.In the BreakOff /Streams window, under the BreakOff tab, turn on the Drop Drive.BreakOff frequency is set at 22.3 Khz.Amplitude at about 3.0 volts. This may need to be slightly increased through theday.When the Drop Drive is turned on, noise may appear in the oscilloscope window. If itlooks like FSC noise, adjust the obscuration bar. If it looks like SSC noise, adjust the twosilver dials directly above the optics block.Michelle has marked the Droplet Screen to indicate the Last Connected Drop (LCD),Gap, and Drop #1. Drop #1 should be very close to Viewing Mark 150. Adjust theBreakoff Amplitude and Breakoff Phase to place LCD and Drop#1 in the proper place Adjust the amplitude to optimize the last connected droplet and minimize thesatellite droplets.Put the charging plates into position, angled out slightly at the bottom.Make sure the stream is going into the center of the waste collection trough.Turn the Plate Voltage to ON on the instrument panel.Do NOT touch the charged plates when the Voltage is on Double check the stream is still going into the center of the waste collectiontrough. If not, adjust with the dial.Turn on the side streams in the BreakOff/Side stream window, using the TestSort button.The side streams can be dragged in or out using numerical values under theStreams Tab.Check that side & center streams all look tight. It may be easier to see if thestream laser is dimmed slightly.Adjust 2nd/3rd & 4th drops if necessary to tighten the center stream.ACCUDROP: Remove rainbow beads from the sample port.Set dial to RUN with no tube in place to rinse the up-take tubing.Open the ACCUDROP experiment in the Browser window.Load a tube of Accudrop beads.RUN.Turn off all external lights and get the room as dark as possible.Run the beads at about 4000 events/sec.Click Acquire on the software.In the sort window, set the precision to Initial.28
FACSCalibur, and FACSVantage-SE SOPJanuary 6, 2011 Put Accudrop filter in place by flipping the small silver bar on the side of the sortdoor.Adjust the Drop Delay to optimize the stream on the left.Set Sort Preciscion to Fine TuneAdjust drop delay again, focusing on the center stream. Try to get the center(waste) stream as dim as possible.VANTAGE SHUT-DOWN: PC: Remove sample tube.Turn off the plate voltage.Turn off the Drop Drive.With Fluidics switch in Standby, hold Nozzle Flush button and rotate Fluidicsfrom Standby to Off.Dissconnect Seath Tank pressure line and feed line, Connect pressure andfeed line to ETOH tank.Turn Fluidics switch to Standby, then to Run (no tube on sample p
Flow Cytometer Sorter (FACS) Calibur and Scanner Standard Operating Procedures document, (2) documentation of sample preparation and transport to the FACS facility, (3) FACS users names and training level. 3.0 Determination of Hazards and Controls 3.1 Hazards The main hazards associated w
