cytof2 sample acquisition: loop system (cytof software version

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.