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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