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29Feb2016SampleSetupandAcquisition
CyTOF2SampleAcquisition:LoopSystem(CyTOFsoftwareversion6.0.626)
ThisprotocolprovidesinstructionsintheeventoneisusingtheinstrumentembeddedloopsystemtoprocesssamplesontheCyTOF.Werecommendusingtheloopsystemforsamplesfromwhichoneiscontentcollectingbetween10,000and200,000datapointspersample(Antibodytitrations,Paneltest,etc).Furthermore,werecommendonlyrunningsamplesusingLoop1inthistwo-loopconfigurationleavingLoop2forwashesbetweensamples.
Objective:ReducescarryoverbetweensampleinjectionsbycompletelyflushingoutthesampleplumbingbetweentheloopsystemandthenebulizerandbycompletelyflushingouttheLoopbeingusedforDatacollection.
Methodassumesyouarestartingfromapointinwhichbothsampleloopshavebeencleaned
A. Beadpreparation(RecommendedthatCalibration/NormalizationbeadsbeaddedtosamplesasaninternalStandard,canjustaseasilybereplacedorsupplementedwithnormalizationbeadsofyourchoosing-EQFourElementCalBeads-Cat#201078:Ce140/142,Eu151/153,Ho165,Lu175/176)
1. NotethatyourpanelMUSTcontainchannelsforCe140,Eu151,Eu153,Ho165,andLu175regardlessoftheirdesignation
2. VigorouslyshakeCyTOFCalibrationBeadsbottle3. Iwillprepareadilutemixture,1:10infreshMilliQnanopurewatereachday
a. Beadswillbeatapproximately3e4beadspermLB. LogIntoyourUseraccountontheWindowsloginpage
1. SUNETUsernameandFACSFacilitypassword2. OpentheCyTOFSoftwarepackagelocatedonthedesktop3. LoopSystem
a. A“Status”WindowwillopenonStartupb. Clickthe“Monitor”tabontheCyTOFhomewindowc. Shrinkbothwindowstoreducefootprintonthephysicalmonitor
29Feb2016SampleSetupandAcquisition
C. SetupAcquisitionParametersandSampleIntroduction
1. Clickthe“Acquisition”IconontheCyTOFhomemenubar2. Rightclickinthe“Gray”Analytetabletoselectapre-existingtemplateortocreatea
newone.
<CRITICAL>MAKEITAHABITTODOUBLECHECKYOURANALYTEPARAMETERS.DATAWILLNOTBESAVEDINEITHERTHE.IMDORTHE.FCSFILEFORMATSWITHOUTBEINGADDEDTOTHEANALYTEACQUSITIONPARAMETERFOREACHEXPERIMENT.<CRITICAL>
3. ForPre-existingtemplates,highlightyourtemplatefromthedatabaselista. Press“Selecttemplate”button
29Feb2016SampleSetupandAcquisition
4. Foranewtemplatea. ScrolldowntothebottomoftheTemplatesdatabaseb. Highlightthe“NewTemplate”entryandenteracustomnameforyourpanelc. ClickthePeriodictableandselectallelementsandthecorrespondingIsotopestobeincludedinyourexperimentalpanel
i. Press“OK”topopulatethetableontheleftd. Press“SelectTemplate”
5. IntheAcquisitionTaboftheAcquisitionwindow,specifyapathwayandfilenametosaveyourdata
a. CyTOFdatashouldbestoredintheE-drivefoldernamed“CyTOF_Data”i. Createauserspecificfoldernamedwithyourinitialsandthedate
6. IntheAcquisitionTaboftheAcquisitionwindow,setupAcquisitionParametersa. Acquisitiontime:dependsofuser
i. Samplesflowrateis~0.045mL/minalong520µLloopvola. 600sec-timenecessarytorecover95%ofacquirabledata
b. AcquisitionandDetectorStabilitydelayi. LoopSystem:30secand10sec
a. Allowtimeforsamplestomakewaytodetector
29Feb2016SampleSetupandAcquisition
7. IntheAnalysisTaboftheAcquisitionwindow,setupdatadefiningparametersa. EnteravalueforMaximum/minimumeventdurationinpushunits
i. Defaultis150/10pushunitsb. Enteravaluefor“TargetEvents”orleaveat0forUnlimited
D. SamplePreparationwithbeads1. Performacellcountoneachsamplepreptodeterminethecellconcentration
a. Finalconcentrationshouldbenolessthan2e5andnomorethan8e5cellspermLinafinalvolumeof600uL
2. The600uLvolumeshouldbecreatedusingthedilutedbeadmixtureprovidedandpreparedeachmorning.
3. Passthecell/beadmixturethroughabluecaptubewitha35ummesha. Collectthecell/beadmixturewitha1mLsyringeensuringatleasta600µLfinalvolumetopreventairintheloopsystem
29Feb2016SampleSetupandAcquisition
4. Sampleintroduction:a. UsetheManualValveSwitchtomakeLoop2theactiveloop.
b. Inject~600µLofsampleintothesampleinjectionport
<Critical>Ensurethatnobubblesremaininthesampleline.<Critical>
5. IntheAcquisitionTaboftheAcquisitionwindow,selectthe“Control”tab
29Feb2016SampleSetupandAcquisition
6. Click“Run”–ThiswillmakeLoop1theActiveSampleloopontheSyringePumpPanel7. OnceCompletedaPlotViewWindowwillopenwithanopportunitytopreviewthedata8. ReturntotheAcquisitionTaboftheAcquisitionWindowtospecifyanewfilenamefor
yournextsample.(SeeSectionC5)E. Cleaningbetweensamples
1. Whilecollectingdata,collect3mLofwaterintoasyringeandinject~0.5mLintoLoop2(CurrentIdleloop).
2. Uponcompletionofdatacollection(acquisitionisstoppedeithermanuallyorbyreachinganeventtime/numberthreshold),usetheManualValveSwitchtomakeLoop2againtheactive.
3. Wait2-4minutesmeasuredmostaccuratelybywaitingforthesyringepumpmetertoreachavolumebetween0.100mLand0.150mL.
4. Duringthis2-4minutewait,inject2.5mLofMilliQwaterintoLoop1(Idleloop)a. ThiswillflushLoop1with5Xvolumewater.
5. PressREPREVIEWtoassessthecleanlinessoftheTimeofFlight.a. Noeventsb. Nobackgroundsignalfromcontaminatedchannels
F. FinalWash1. Whilecollectingdataforthefinalsample,collect3mLofHFWashSolutionintoa
syringeandinject~0.5mLintoLoop2(CurrentIdleloop).2. Uponcompletionofdatacollection(acquisitionisstoppedeithermanuallyorby
reachinganeventtime/numberthreshold),usetheManualValveSwitchtomakeLoop2againtheactive.
3. Wait2-4minutesmeasuredmostaccuratelybywaitingforthesyringepumpmetertoreachavolumebetween0.100mLand0.150mL.
4. Duringthis2-4minutewait,inject2.5mLofHFWashSolutionintoLoop1(Idleloop)
29Feb2016SampleSetupandAcquisition
a. ThiswillflushLoop1with5Xvolumewashsolution.5. Collect3mLofMilliQwaterintoasyringeandinject~0.5mLintoLoop2(CurrentIdle
loop).6. UsetheManualValveSwitchtomakeLoop2againtheactive.7. Wait2-4minutesmeasuredmostaccuratelybywaitingforthesyringepumpmeterto
reachavolumebetween0.100mLand0.150mL.8. Duringthis2-4minutewait,inject2.5mLofMilliQwaterintoLoop1(Idleloop)
a. ThiswillflushLoop1with5XvolumeMilliQwater.9. PressREPREVIEWtoassessthecleanlinessoftheTimeofFlight.
a. Noeventsb. Nobackgroundsignalfromcontaminatedchannels
29Feb2016SampleSetupandAcquisition
DataCollectionattheEndoftheDay
G. Normalization1. OpentheFCSAnalysisTabonthetoppaneloftheCyTOFsoftware2. UsetheBrowsebuttontoloadfilesintothe“SourceFile”3. UndertheNormalizationFeatureselecttheBeadLotassociatedwiththebeadsusedin
yoursamplefromthe“NormalizationBeads”pulldownwindow4. PresstheStartButtonatthebottomoftheFCSAnalysisWindow5. TheOutputfilescanbefoundinthefoldercontainingyourSourcefileseachannotated
withanadditional“_1”attheendofthefilename.
H. DataCollection(TheFACSfacilityisaUSBflashdrivefreezone)
1. MinimizetheCyTOFsoftware2. TransferyourdatafilesfromtheE:DrivetotheC:Drivefolder“CyTOFdata”3. Open“FACSDataCheckIn”foundontheDesktop4. AWindowwillopenaskingyouto“SelectanExperiment”
a. ThedatafolderyoucreatedinPartC3aiwillbehighlightedb. SelectandPressOK
5. Awindowwillopenaskingyouto“Selectaninvestigator”a. Yourlastandfirstnameshouldappearatthetopb. SelectandPressOK
6. AlastwindowwillenableyoutoaddadditionalemailaddressestotheFACSDataCheckInalertsystem
a. PressOK
29Feb2016SampleSetupandAcquisition
7. Youwillbepromptedthatdatawillbeavailablefromaremoteservershortlya. Datatakesapproximately15minutestotransferb. Anemailwillpromptyoutothecompletionofthetransfer
I. LogOut(ShouldtherebeaUserafteryou)1. CloseCyTOF(andallotherprogramsthatmaybeopenonthedesktop2. LogOutofyourwindowsaccounttoendthatday’saccounting
J. Retrievingdata(Fromanywhere,worldwide)1. GototheSharedFACSFacilitywebsite:http://facs.stanford.edu
2. ExpandfromtheleftframetheDataManagementheadertorevealtheDataArchivelink
a. ClicktheDataArchivelink
b. WhenPromptedenteryourSUNETIDandpassword
i. You’llneedaWebAuthenticatortoproceed
c. WhenpromptedentertheUsername:flowjo,Password:314159
3. YoushouldnowhaveaccesstoallyourgeneratedFCSfilesfromtoday’ssession
29Feb2016SampleSetupandAcquisition
Intheeventofaplasmafailure
K. RestartingthePlasma1. IfinthemidstofanAcquisitionpleasePressSTOPintheAcquisitionwindow“Control”
tab.Doingthiswillsaveyourdatafileforconcatenationlater.2. Doamanualvalveswitchtoensureyoursampleisintheinactiveloop(Loop2).3. Clickthe“About”TabontheCyTOFhomewindow
a. Select“Login”intoadministratoruseraccounti. Enablesaccesstothe“ControlPanel”ii. Administrator/donuts
4. Clickthe“ControlPanel”TabintheCyTOFMainMenutoopenthecontrolpanel5. Clickthe“Plasma”TabtoopenthePlasmacontrol6. Click“StartPlasma”
a. PlasmaStartuptakesabout5minutesL. ResumeAcquisitionasdescribedinPartC5
1. Notethatyouwillneedtopress“Run”intheacquisitionwindow’scontroltabtoreturntotheloopcontainingyoursample.
Askforhelpifyouhaveanyquestions,Brandon,cell(775)7224672.