2018 principles of flow cytometry 2018 maria daly web€¦ · the university of chicago flow...
TRANSCRIPT
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FlowCytometry
MariaDalyFlowCytometryFacilityManagerMRC-LMB
TalkOverview
-OurFacility- WhatisFlowCytometry?- ComponentsofaFlowCytometer- ApplicaHonsinBiology
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OurFacility:thePeople
FanZhang MartynBalmontMariaDaly
OurFacilityattheLMB
MoFloHighSpeedSorter:• 4lasers,8colourdetecHon
SynergydualchannelHighSpeedSorter:1) 5lasers,15colourdetecHon2) 3lasers,10colourdetecHon
LSRIIAnalyser:4lasers,14colourdetecHon
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OurFacilityattheLMB
2xFortessaAnalysers:1. 4lasers,16colourdetecHon2. 5lasers,18colourdetecHon
1xSonySP6800SpectralAnalyser:Thisisa3laser,34PMTdetecHonsystemwhichcapturesthespectralfingerprintofeachfluorochrome
• 405nm,50mWlaser,with2PMTdetectors(420-440,460-480nm)• 488nm,50mWlaser• 640nm,40mWlaser
Emissionspectrafromabove3excitaHonlasersisdetectedacrossabandof32fluorescencedetectorsdetecHngemissionwavelengthsfrom500–800nm
OurFacilityattheLMB
EclipseAnalyser:• 3lasers,5colourdetecHon
FACSCaliburAnalysers:• 2lasers,4colourdetecHon
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PublicaHonssupportedbytheFacilityfromOctober2016–September2017
GammonsM.V.,RutherfordT.J.,SteinhartZ.,AngersS.,BienzM.EssenHalroleoftheDishevelledDEPdomaininaWnt-dependenthuman-cell-basedcomplementaHonassayJournalofCellScience129(20):3892-3902.15October2016Division:Protein&NucleicAcidChemistryBoQermannM.,LodeH.E.,WatkinsonR.E.,FossS.,SandlieI.,AndersenJ.T.,JamesL.C.AnHbody-anHgenkineHcsconstrainintracellularhumoralimmunityScien?ficReports6:37457.24November2016Division:Protein&NucleicAcidChemistryWuY.L.,StubbingtonM.J.,DalyM.,TeichmannS.A.,RadaC.IntrinsictranscripHonalheterogeneityinBcellscontrolsearlyclassswitchingtoIgEJournalofExperimentalMedicine214(1):183-196.1January2017Division:Protein&NucleicAcidChemistryMcEwanW.A.,FalconB.,VaysburdM.,CliWD.,OblakA.L.,GheYB.,GoedertM.,JamesL.C.CytosolicFcreceptorTRIM21inhibitsseededtauaggregaHonProceedingsoftheNa?onalAcademyofSciencesoftheUnitedStatesofAmerica114(3):574-579.17January2017Division:Protein&NucleicAcidChemistry,NeurobiologySaluzzoS.,GorkiA.D.,RanaB.M.,MarZnsR.,ScanlonS.,StarklP.,LakovitsK.,HladikA.,KorosecA.,SharifO.,WarszawskaJ.M.,JolinH.,MesteriI.,McKenzieA.N.,KnappS.First-Breath-InducedType2PathwaysShapetheLungImmuneEnvironmentCellReports18(8):1893-1905.21February2017Division:Protein&NucleicAcidChemistryvanTienenL.M.,MieszczanekJ.,FiedlerM.,RutherfordT.J.,BienzM.ConsHtuHvescaffoldingofmulHpleWntenhanceosomecomponentsbyLegless/BCL9ELife6:e20882.15March2017Division:Protein&NucleicAcidChemistryHoulihanG.,Arangundy-FranklinS.,HolligerP.ExploringtheChemistryofGeneHcInformaHonStorageandPropagaHonthroughPolymeraseEngineeringAccountsofChemicalResearch50(4):1079-1087.18April2017Division:Protein&NucleicAcidChemistryŠvikovićS.,SaleJ.E.TheEffectsofReplicaHonStressonSPhaseHistoneManagementandEpigeneHcMemoryJournalofMolecularBiology429(13):2011-2029.20June2017Division:Protein&NucleicAcidChemistryBurgos-BarraganG.,WitN.,MeiserJ.,DinglerF.A.,PietzkeM.,MulderrigL.,PontelL.B.,RosadoI.V.,BrewerT.F.,CordellR.L.,MonksP.S.,ChangC.J.,VazquezA.,PatelK.J.Mammalsdivertendogenousgenotoxicformaldehydeintoone-carbonmetabolismNature548(7669):549-554.31August2017Division:Protein&NucleicAcidChemistry
Thevalueofthetechnique:• measurementsoflargenumbersofsinglecells
insuspensionwithinashortperiodofHme
Themajordisadvantage:• Itrequiresasuspensionofsinglecellsorother
parHcleswithminimumclumpsanddebris• TheHssuearchitectureandanyinformaHonabout
thespaHalrelaHonshipbetweendifferentcellsarelostwhensinglecellsareprepared
FlowCytometry
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Cytometryl LocalizaHonofanHgenis
possiblel PoorenumeraHonofcell
subtypesl Tissuearchitecture
FlowCytometryl Cannottellyouwhere
anHgenisl Cananalyzemanycellsin
ashortHmeframel Canlookatnumerous
parametersatonceDAPI IL-13eGFP CD3IL-25; 3 days; i.n.
CD4
IL-13eGF
P
JillianBarlowandVeeraPanova,MRC-LMB
ComponentsofaFlowCytometer
hhp://www.thermofisher.com/uk/en/home/support/tutorials.html
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CellscomeinavarietyofdifferentsizesMakesuretheywillfittheflowcellornozzleyouuse
SamplePreparaHon
• SamplepreparaHoniskeytogeinggooddata• Asinglecellsuspensionisnecessary• Dissociatecellswithappropriatereagents• TitrateyouranHbodiestofindopHmalconc.• Filtercellsampleswhichareaggregatedthroughanylonmeshto
removeclumps• 70μm,100μm,or150μmasappropriate
• Rubbishin=Rubbishout
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Obtainingasinglecellsuspension
• Somecellscomenaturallyinsuspension• splenocytes• celllinese.g.Jurkat,
• SomecelllinesgrowadherentonplasHc• removefromplasHcwithe.g.EDTA,Trypsin,Accutase
• TissuesaremoredifficultandneedmechanicalorenzymaHcdissociaHon• Collagenase• Pressthroughafinemesh
• Thesampleshouldcontainaslihledebrisandasfewclumpsaspossible
Hydrodynamicfocusing
SingleParHclesinastream
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ForwardScaher
ForwardScaher
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ForwardScaherPulses
ForwardScaherHistogram
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Threshold
SideScaher
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SideScaher
2ParameterDotPlot
ForwardScaher
SideScaher
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WholeBloodDotPlot
OpHcalLayout
FluorescencedetecHon
ForwardScaher
SideScaher488nmlaser
Dichroicmirrors
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FiltersinfrontofdetectorstoseparateFluorescenceλ
So,runsinglecolourcontrolstoassessthespectraloverlap
Fluorescence
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FluorescenceHistogram
FluorescenceDotPlot
SS
CD45-ECDLYMPHS
PBMC
WBC
FreshWholeBlood
CD4-PC7
CD8-FITC
CD8-FITC
CD4-PC7
CD45-ECD
CD45-ECD
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OpHcalLayout
FluorescencedetecHon
ForwardScaher
SideScaher488nmlaser
Computer
• Avoidco-incidence• DilutethesampletoaconcentraHontheinstrumentcanhandle
• Getridofunwantedcells• LyseRBC
• AmmoniumChloride• RBClysingreagentse.gacidlysis• Carbonicanhydrase
• FicollorLympholyteM• SeparatesmononuclearcellsfromGranulocytesandRBC
OtherconsideraHons
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Alwaysincludecontrols
• TheinstrumentseingsforcollecHngcellmeasurementsaresetbyyou
• Usecontrolstosetyourbackgroundvalues• Usesinglecolour-samplestosetyourcolour
compensaHonandposiHonofposiHvecells• Choosedyesusedinyourexperimentswisely
ü brightestdyeonleastexpressedanHgenü choosedyeswithleastspectraloverlapor
whichusedifferentlasersforexcitaHon
LabellingCellswithFluorescentDye/Marker
• OtpimisethestainingcondiHonsforYOURcellsinyourmodelsystem
• Immunophenotyping• DNAanalysis• IntracellularCytokineStaining• CFSEproliferaHonassay
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Immunophenotyping
Protocol:-100μlsample-10μlanHbody-10minsRT- WashwithPBS+2.5%FCS- Centrifugetopellet,removesupernatant- Re-suspendinPBS+2.5%FCS-Analyse
Immunophenotyping
• Readtheproductinsertsheet
• FindtheconcentraHonofthereagente.g.mganHbody/ml• ReadoffrangeofanHbodyconcentraHonmanufacturer
recommendsforuse
• TitrateyouranHbodiestofindtheopHmal
concentraHonforuse• Keepfinalvolumeconstante.g.100μl• AnHbodyconcentraHonisvital,cellnumbersmayvary• AddrangeofanHbodyconcentraHonatconstantvolume• FindsaturaHngconcentraHonforuse
• Beconsistent
• UsesamestainingcondiHonseachHmeyoudotheexperiment
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AnHbodyTitraHon
n Formostpurposes,themainobjecHveistomaximizesignal:noise(pos/negseparaHon)n Thismayoccuratlessthansaturatedstainingn Thismayormaynotbethemanufacturer’srecommendedHter
n Titerisaffectedby:n Stainingvolume(e.g.,100µL)n Numberofcells(notcriHcalupto~5x106)n StainingHmeandtemperature(e.g.,10minRT)n Typeofsample(wholeblood,PBMC,splenocytes.)
AnHbodyTitraHon
TheUniversityofChicagoFlowCytometryFacility
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AnHbodyTitraHon
0
2000
4000
6000
8000
10000
12000
10 3.3 1.1 0.37 0.12 0.04 0.0140.005
Noise
Signal
0
10
20
30
40
50
60
70
10 3.3 1.1 0.37 0.12 0.04 0.014 0.005
RaHo
ConcentraHonμg/ml
Med
ianFluo
rescen
ce
OpHmal
ConcentraHonμg/ml
Signal/N
oise
Immunophenotyping
LYMPHS
SS
CD45-ECD
PBMC
WBC
FreshWholeBlood
CD8
CD3
CD8
CD4 CD25
CD4
CD19
HLA-DR
HLA-DR
CD95
CD11
c
CD56Protocol:-100μlsample-10μlanHbody- 10minsRT- RBClyse-Wash-Analyse
TitrateanHbodiestofindopHmalconcentraHonforuse
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BeCarefulofReagentDegradaHon!
CD19
PEC
y7
CD3PE
Lightwillphotobleachyourtandem–protectyouranHbody-labelledsamplesfromlightatallHmes
PE488nmor561nmlaserexcitaHon
575nmemissionoutandabsorbed
Cy7780nmemihed
Sample NextSample LaterSample EvenLaterSample
≈
BeCarefulofReagentDegradaHon!
CD19
PEC
y7
CD3PE
Lightwillphotobleachyourtandem–protectyouranHbody-labelledsamplesfromlightatallHmes
PE488nmor561nmlaserexcitaHon
575nmemissionout
Sample NextSample LaterSample EvenLaterSample
≈
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PEspectrum PECy7(degraded)spectrum
SpectralAnalyser
WecananalysethespectrumofFluorochromesandrevealthedegradaHon
Emissionwavelengthinηm
CellCycleAnalysisAnumberofdyescanbeusede.g.-PropidiumIodide-Hoechst-DAPI- DRAQ5
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CellCycleAnalysis
Eve
nts
Forw
ard
Sca
tter P
ulse
Are
a
Hoechst λex = 405ηm λem = 450ηm
Singlets
Clumps Haploid Diploid
Theselection of single cells Histogram of DNA profile
Data from Ben Taylor (PNAC), MRC-LMB
Forward Scatter, Pulse Height
CellCycleAnalysis
HoechststainingofViablecells
Singlets
GateonSinglets
EmmanuelBoucrot
HoechstIntergral
HoechstPeak
Hoechst
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R6
R7
0 64 128 192 256FL8 450integral
100
101
102
103
104
FL1 GFP Comp
CellCyclewithGFP
IvanRosadoPNAC,MRC-LMB
Hoechst
GFP
0 64 128 192 256FL8 450integral
0
211
423
635
847
Counts
0 64 128 192 256FL8 450integral
0
135
270
405
540
Counts
Hoechst
Hoechst
HoechststainingcombinedwithGFPNofixaHonrequiredthusleavingGFPexpressionunaffected
CellCycle
FindtheopHmalstainingconcentraHonforyourcellsKeepcellconcentraHonandTIMEofstainingatrequiredtemperatureconsistentbetweenexperiments!!DisaggregatepopulaHonMinimizeclumps
R2
0 64 128 192 256FL6 450peak
0
64
128
192
256
FL8 450integral
0 64 128 192 256FL8 450integral
0
115
230
345
460
Counts
HoechstHoechstPeak
HoechstIntegral
43.1%
Sub-opHmalstaining
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ProliferaHonusingCFSE
Protocol:-WashsamplewithPBS,noprotein-Re-suspendcellsin5uMCFSE-10minsat37oC-Wash- Culture- Analyse
As the CFSE loaded cell divides, the fluorescence is halved
Log FL1
Log FL1
ProliferaHonusingCFSE
- CellTraceVioletCellProliferaHonKit(Invitrogen)- CellProliferaHonDyeeFluor670(eBioscience)
AlternaHvecolours:
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CFSE
PBMC
Measurement of Cell Division and Phenotype
Setupincultureandmonitorover7daystolookatresponse
CD4-PC7
+SHmulantCD8-ECD
CFSE
HumanPBMC,Day7
CFSE
UnsHmulated:
CFSE
+SEB:
CD4-PC
7
CD8-PC
5
CD4
CD8CFSECFSE
CD4
CD8-PC
5
CD4-PC
7
CD8CFSECFSE
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Channels (FL1 Log-CFSE-FL1 Log)0 50 100 150 200 250
Num
ber
070
140
210
280
ProliferaHonWizardBasicModelFile:10_m4aSpecimenR_100000382010.LMDDateacquired:16-Feb-06Dateanalyzed:22-Feb-2006Parent:9.49%at234.00GeneraHon2:6.83%at212.13GeneraHon3:6.10%at189.93GeneraHon4:9.66%at169.90GeneraHon5:14.06%at150.82GeneraHon6:19.16%at132.43GeneraHon7:17.65%at115.96GeneraHon8:12.05%at100.68GeneraHon9:4.46%at86.61GeneraHon10:0.55%at71.26ProliferaHonIndex:5.71NonproliferaHveFracHon:0.54DivisionErrorIndex:1.00SpacingofgeneraHons:21.87ForcellsatgeneraHon>=3:UpperGeneraHonP.I.:18.20PrecursorFrequency:0.262734NumberofCellsAnalyzed:23443ReducedChi-Square:1.837
ParentGeneration 2Generation 3Generation 4Generation 5Generation 6Generation 7Generation 8Generation 9Generation 10
Culture4:(55928-CFSE+PMA+Ionomycin),
ExamplecalculaZonofPrecursorFrequency(Modfit)
Channels (FL1 Log-CFSE-FL1 Log)0 50 100 150 200 250
Num
ber
030
060
090
012
00
ProliferaHonWizardBasicModelFile:01_m1aSpecimenR_100000373001.LMDDateacquired:16-Feb-06Dateanalyzed:22-Feb-2006Parent:97.22%at224.00GeneraHon2:1.95%at204.81GeneraHon3:0.23%at185.62GeneraHon4:0.14%at166.43GeneraHon5:0.08%at147.24GeneraHon6:0.16%at128.05GeneraHon7:0.11%at108.86GeneraHon8:0.06%at89.67GeneraHon9:0.04%at70.48GeneraHon10:0.01%at51.29ProliferaHonIndex:1.02NonproliferaHveFracHon:0.99DivisionErrorIndex:1.00SpacingofgeneraHons:19.19ForcellsatgeneraHon>=3:UpperGeneraHonP.I.:9.43PrecursorFrequency:0.000894NumberofCellsAnalyzed:19175ReducedChi-Square:7.583
ParentGeneration 2Generation 3Generation 4Generation 5Generation 6Generation 7Generation 8Generation 9Generation 10
Culture1:(55928-CFSE),noresHmulaHon
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IntracellularCytokineStainingProtocol:-Pelletcells(1to3x106)-AddIntraPrepFixaHve-Incubate15minsRT-Wash- AddIntraPrepPermeabilizaHonreagent- AddappropriateanHbodyconcentraHon- Incubate15minsRT- Wash- Analyse
Fix
15minsWash
Permeabilize
WashAnalyse
Addbuffer
+anHbodies15mins
IntracellularCytokineStainingMethod:
1x106Tcells+1x105APC±sHmulant
6hrs.@370C
4hrsMonensinorBrefeldinA
wash
Addα-CD810mins.,RT.
wash
AddfixaHvewash
AddpermeabilizaHonagent+anHbodies,15mins.RT.
AnalyseonFlowCytometer
wash
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CFSEandImmunophenotypeandIntracellularCytokines
APC-IFNγ
PE-IL-2
CD8-ECD
APC-IFNγ
PE-IL-2
CD4-PC7
5-ColourCombinaZonwithdualCytokine
CFSE
CFSE
+PMAandIonomycin:
A1 A2 A3 A4
B1 B2 B3 B4
CFSECFSE IFNγ
IL-2
IL-2
IFNγ
CFSECFSE IFNγ
IFNγ
IL-2
IL-2
IL-2producedfromfirstcelldivisiononwardsIFNγproduceda|eranumberofcelldivisions
Day7+/-sHmulaHonUnsHmulated:
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SorHng
MoFloCellSorter
Streamview
Thestreamisvibratedathighfrequencysothatdropletsareformed
UpperChamber
Lasersinterceptsamplestream
Dropletscanbechargedeither- PosiHve+- NegaHve-
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OurMoFloCellSorter
-2500V+2500V
Wastecatcher
Cellsindropletspassthroughchargedplates
Wastecatcher
-2500V+2500V
MoFloCellSorter
UpperChamber
Lasersinterceptsamplestream
LowerChamber
StreamdropletspassdownthroughchargedplatesanddeflectedintocollecHontubesorplate
-2500V+2500V
Wastecatcher
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Sortintotubes,plates,whatever
-2500V
WastecatcherPurifythepopulaHonyouwantbasedonfluorescenceandlightscaher
ReferenceBooks:• PracHcalFlowCytometry.HowardMSharpiro(freepdf)• FlowCytometryAPracHcalApproach.MGOrmerod• CytometricAnalysisofCellPhenotypeandFuncHon.McCarthy&Macey• IntroducHontoFlowCytometry.JVWatson.
WebReferences:• FLOWCYTOMETRYAbasicintroducHon.MichaelG. Ormerod hhp://flowbook.denovoso|ware.com/
References
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History1965:L.Kamentsky(IBMLabs)andM.Fulwyler(LosAlamosNatLab)experimentedwithfluidicswitchingandelectrostaHccellsorters.1967:MackFulwylerpurified(>95%)bloodgranulocytesandlymphocytesbypassingcellsthroughaCoulterorifice,thenbreakingthestreamintodropletswhichcouldbecharged,andthendeflectedintoacollecHonvesselastheypassedbetweenvoltageplates1967:KamentskybuilttheRapidCellSpectrophotometer(RCS)asyringepumpbasedsorterwhichmeasurednucleicacidcontentofcervicalcellsandcellssizebylightscaher1968:Firstfluorescence-basedflowcytometrydevice(ICP11)wasdevelopedbyWolfgangGöhde.CommercializedbyGermandeveloperandmanufacturerPartecthroughPhyweAGinGöingen.ThetechnologywastermedPulseCytophotometry.PartecwasacquiredbySysmexin20131969:EthidiumbromidefirstusedbyDihrichandGöhde1971:TheCytofluorographfromBio/PhysiceSystemsInc(laterOrthoDiagnosHcs)1972:L.Herzenberg(Stanford)developedacellsorterwhichseparatedcellsstainedwithfluorescentanHbodies.1973:CrissmanandSteinkampintroducedPropidiumIodide
Flow Cytometry on the web
History.1973:CrissmanandSteinkampintroducedPropidiumIodide1973:PAS8000fromPartec1974:firstFACSinstrumentfromBectonDickinson1974:firstcommercialflowcytometricdifferenHalbloodcounter(HemalogD)1975:theICP22fromPartec/Phywe1976HoechstdyesintroducedbyLahandStehen1977/78:TheEpicsfromCoulter1977:StohrintroducedDAPI1978:ThetermFlowCytometrywascoinedattheConferenceoftheAmericanEngineeringFoundaHoninPensacola,Florida
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Flow Cytometry on the web
GrowthinFlowCytometricStudiesMarch5th2010:PubMedsearchon
"flowcytometry" :107,139citaHons(March5th2010) :179,485citaHons(March8th2016)"cytometry" :110,489citaHons(March5th2010) :184,309citaHons(March8th2016)
FirstcitaHononbothlists:1974paperbyMackFulwylerTheSocietyforAnalyHcalCytology,foundedin1978,establishedCytometryasitsjournalHtlefromthebeginning,butonlyrecentlychangeditsnametotheInternaHonalSocietyfortheAdvancementofCytometry.
1974 2017