F lo w C y to m e t r y

New applications for Vybrant DyeCycle stains

IDeNtIfyINg stem Cell sIDe populatIoNs aND Cell CyClebaseD sortINg.

Live-cell studies of cellular DNA content and cell cycle distribution areusefultodetectvariationsofgrowthpatternsduetoavarietyof physical,chemical,orbiologicalmeans,tomonitorapoptosis,andto studytumorbehaviorandsuppressorgenemechanisms.TheVybrant DyeCyclestains,availableasVybrantDyeCycleVioletstain,Vybrant DyeCycle Green stain, andVybrant DyeCycle Orange stain, were designedtoreportDNAcontentinlivingcells.AllVybrantDyeCycle stainsareDNAselective,cellmembranepermeant,andnonfluorescent untilboundtodouble-strandedDNA.Thesedyestakeadvantageof thecommonlyavailable488nmandvioletexcitationsources,placing cellcyclestudieswithinreachofallflowcytometrists. Recent studies have shown the utility of these dyes in two new applicationareas:

WithDyeCycleVioletstaininmousehematopoieticcells,sidepopulationssimilartothoseobservedwithHoechst33342canberesolved using either violet or UV excitation (Figure 1). Fumitremorgin C, an ABCG2-specificinhibitor,blockedtheappearanceofthiscellpopulation.Furthercharacterizationofthecellsbyimmunophenotypingusing mousebonemarrowstemcellmarkersconfirmedthattheidentified DyeCycleVioletstainSPisrestrictedtothestemcellLSKpopulation (Lineagenegative Sca-1positive c-kitpositive), similar to the Hoechst 33342 SP. TheseresultsstronglysuggestthatDyeCycleVioletstaineffluxidentifiedthesamestemcellpopulationasHoechst33342efflux.*SPanalysis on flow cytometers equipped with violet lasers should therefore be possiblebysubstitutingDyeCycleVioletstainforHoechst33342.
* Fordetailsofthisexperiment,visitwww.invitrogen.com/flowcytometry andfollowthelinkundertheposterentitledCellCycleAnalysisinLiveCells UsingNovelVybrantDyeCycleStains.

dentificationofstemcellsidepopulations(SP)1(Figure1) i ortingpotentialbasedoncellcyclephase(Figure2) s

Cell sorting using Vybrant DyeCycle stains

Flow cytometry testing is useful for quantifying the distribution

Identification of Hoechst side population using DyeCycle Violet stain

Hoechst33342iswidelyusedasareagenttoidentifystemcellsand earlyprogenitorsinmammalianhematopoietictissues.Afterloadinga cellpopulationwithHoechst33342,acell-permeantnucleicacidstain, thesidepopulation(SP)containingstemcellsandearlyprogenitorsis identifiedinthecytometerasalow-fluorescencetail(arisingasthe dyeispumpedoutofthecellviaanABCG2membranepumpdependentmechanism).Thistechniquerequiresaflowcytometerequipped withaUVlaser,anuncommonandrelativelycostlyoption.Asviolet diodelasersbecomemoreprevalentsecondaryexcitationsourceson flowcytometers,theopportunityarisesforperformingthistechnique usingavioletlaserexcitedcell-permeantnucleicacidstain.DyeCycle VioletstainexhibitsthesamepumpspecificityasHoechst33342while beingexcitedbythevioletlaser.

of cell populations across the different nuclear phases of the cell cycle. Analysis of the cell cycle is widely used in cell growth and cell cycle regulation studies, oncology research, and DNA ploidy determinations.TheseapplicationsrequiredyesthatbindtoDNAina stoichiometricmanner.WiththeexceptionsofUV-exciteddyessuch asHoechst33342,cellshavegenerallyrequiredfixationandpermeabilizationaswellastreatmentwithRNasetoobtainDNA-specificcell cycleinformation.TheVybrantDyeCyclestainsareDNA-selective,cell membranepermeantdyesthatshowgreatlyenhancedfluorescence whenboundtoDNAandareavailableinversionsthatcanbeexcited by 405 nm, 488 nm, or 532 nm lasers. Cell cycle analysis using the VybrantDyeCyclestainshasbeenperformedonawidevarietyof cells,includingJurkat,CHO,3T3,andHL60cells,andperipheralblood lymphocytes,monocytes,andneutrophils.

28 | BioProbes52 | March2007

2007InvitrogenCorporation.Allrightsreserved.TheseproductsmaybecoveredbyoneormoreLimitedUseLabelLicenses(seeInvitrogencatalogorwww.invitrogen.com).Byuseoftheseproducts youacceptthetermsandconditionsofallapplicableLimitedUseLabelLicenses.Forresearchuseonly.Notintendedforanyanimalorhumantherapeuticordiagnosticuse,unlessotherwisestated.

DyeCycle Violet blue fluorescence

800

DyeCycle Violet blue fluorescence

1,000

1,000

800

600

600

400

400

200

200

200

400

600

800

1,000

200

400

600

800

1,000

DyeCycle Violet red fluorescence

DyeCycle Violet red fluorescence

Figure 1DyeCycle Violet stain side population analysis in human cord blood. Cellswereincubatedwith10MDyeCycleVioletstainfor90minutesat37C,thenwashedandstoredoniceuntil analysis.ResultswerethesameusingUVexcitation(A)orvioletdiodelaserexcitation(B).Datacourtesy ofWilliamTelford,NIH.

BecauseDyeCycledyescanbeusedforcell cycle analysis on living cells, experiments

Cell count

250

200

wereperformedtodeterminewhetherthese dyes could be used to sort cell populations basedontheirpositionwithinthecellcycle. HEK and NIH3T3 cells were stained with DyeCycle Violet and DyeCycle Orange stains, respectively, then sorted using a BD FACSVantage flow cytometer. Figure 2
Cell count
250

150

100 50 G1 G2 0 50 100 150 200 250

G0/G1
25

G2/M

200

20

Cell count

shows NIH3T3 cells stained with DyeCycle Orangestain.WhiletheVybrantDyeCycle stainscausedsomeretardationofcelldivision, they did not produce the toxicity reported withDRAQ5stain(BiostatusLtd.)andhave beenusedtosortviablecellsfromG0/G1and G2/M populations. Resolution of cell cycle information in viable cells allows evaluation againstthedynamicbackgroundoflive-cell activity,aswellasthepossibilityofcellsorting basedonpositioninthecellcycle.

150

G0/G1

G2/M

15

G0/G1

G2/M

100 50 0

10 5

50

100

150

200

250

50

100

150

200

250

Vybrant DyeCycle Orange

Vybrant DyeCycle Orange

3 days post-sort

Reference
1. Telford, William G. et al. (2007) Stem Cells (inpress).

Figure 2Sorting of live-cell populations. NIH3T3 cells were stained with 10 MVybrant DyeCycleOrangestain.CellsweresortedbasedonG0/G1andG2/MgatesusingaFACSVantage flowcytometer(BDBiosciences)with488nmexcitationanda585/42nmbandpassfilter.Cells wereculturedaftersorting.

Product
VybrantDyeCycleVioletstain*5mMinwater**200assays* VybrantDyeCycleGreenstain*5mMsolutioninDMSO**200assays* VybrantDyeCycleOrangestain*5mMsolutioninDMSO**200assays*

excitation
UV,405nm 488nm 488nm,532nm

Quantity
200l 400l 400l

Cat. no.
V35003 V35004 V35005

2007InvitrogenCorporation.Allrightsreserved.TheseproductsmaybecoveredbyoneormoreLimitedUseLabelLicenses(seeInvitrogencatalogorwww.invitrogen.com).Byuseoftheseproducts youacceptthetermsandconditionsofallapplicableLimitedUseLabelLicenses.Forresearchuseonly.Notintendedforanyanimalorhumantherapeuticordiagnosticuse,unlessotherwisestated.

probes.invitrogen.com | 29

F lo w C y to m e t r y

JourNal HIgHlIgHt

A new approach to cell-based multiplexing expands the drug screening capabilities of flow cytometry
Krutzik,P.O.andNolan,G.P.(2006)FluorescentCellBarcodinginFlowCytometryAllowsHigh-ThroughputDrugScreeningand SignalingProfiling.Nature Methods5:361368.

Can high-throughput, high-content flow cytometry be economically applied to large-scale drug screening?Flowcytometryisawidely utilizedandpowerfulmethodfortheanalysisofmultipleantigens incellpopulations.However,theuseofflowcytometryindrug screeningapplications,whichcaninvolvehundredsorthousands ofsamples,canquicklybecomecostandtimeprohibitive,dueto theamountsofantibodiesrequiredandthethroughputlimitationsofcytometers.Theauthorspresentacell-basedmultiplexing approachfluorescentcellbarcoding(FCB)thatusesvaried stainingintensitiestoallowtheanalysisofcomplexsamplesin asingleflowcytometryrun. Inthistechnique(basedonstandardphosphoflowprotocols),cellsamplesthathaveundergoneaninitialtreatment (e.g.,unstimulated,stimulated,andstimulatedinthepresence ofaninhibitordrugcandidate)arebarcodedbygeneralstainingwithdifferentlevelsofareactivefluorophore(PacificBlue, Alexa Fluor 488, Alexa Fluor 700, and Alexa Fluor 750 fluorophoreswereallshowntobeeffectivebarcodingdyes). Followingbarcoding,thesamplesarethenrecombined,stained withfluorescentlylabeledantibodiestodetecttheeffectsof thetreatment,andanalyzedasasinglesample.Deconvolution oftheresultsclearlyresolvesthedifferentiallytreatedcellsinto discrete, quantifiable populations.The authors successfully demonstrate the utility of the method for real-world drug

screening applications. In an inhibitor-titration experiment usingthePacificBluefluorophoreasthebarcodingdye,U937 monocytecellswerepretreatedwithfoursmall-moleculeinhibitorsofJAKkinases,thenstimulatedtoinducepStat1,pStat3,and pStat5production.Theeffectoftheseinhibitorsonthedegree ofphosphorylationofthethreeStattranscriptionfactorswas clearlyrevealedinasingleflowcytometryrun. In a separate experimenta 96-well platebased drug candidatescreeningapplicationtheauthorsemployedathreedyeFCBbarcodingschemetolabel96samples.Theyusedthis schemetoscreenalibraryof70small-moleculeinhibitorsfortheir effectonTcellreceptormediatedERKphosphorylation/Stat1 phosphorylationinresponsetointerferon-(IFN-)treatment. Thisscreeningexperimentwascompletedinasingle5minute flowcytometryrun,andidentifiedtwocompoundsthatselectivelyinhibitedoneortheotherpathwayandthreecompounds thatnonselectivelyaffectedbothpathways. Overall, the authors report up to 100-fold reduction in antibodyconsumption,withsignificantlylessacquisitiontime requiredforcomplexsampleanalyses.Owingtoitsimproved throughputandgreatlyreducedconsumptionofantibodies,the FCBmethodologymayproveusefulfordrugcandidatescreeningaswellasforclinicalmonitoringofpatientsamplesduring late-stagedrugtrials.

Product
AlexaFluor488carboxylicacid,succinimidylester*mixedisomers* AlexaFluor488carboxylicacid,succinimidylester*mixedisomers* AlexaFluor700carboxylicacid,succinimidylester*mixedisomers* AlexaFluor700carboxylicacid,succinimidylester*mixedisomers* AlexaFluor750carboxylicacid,succinimidylester*mixedisomers* AlexaFluor750carboxylicacid,succinimidylester*mixedisomers* PacificBluesuccinimidylester

Quantity
1mg 5mg 1mg 5mg 1mg 5mg 5mg

Cat. no.
A20000 A20100 A20010 A20110 A20011 A20111 P10163

30 | BioProbes52 | March2007

2007InvitrogenCorporation.Allrightsreserved.TheseproductsmaybecoveredbyoneormoreLimitedUseLabelLicenses(seeInvitrogencatalogorwww.invitrogen.com).Byuseoftheseproducts youacceptthetermsandconditionsofallapplicableLimitedUseLabelLicenses.Forresearchuseonly.Notintendedforanyanimalorhumantherapeuticordiagnosticuse,unlessotherwisestated.

Stimulate and treat with drug

Fix with formaldehyde

Standard phospho flow

Permeabilize with methanol

FCB phospho flow

Permeabilize with methanol, add FCB markers

Cell number
102

103

104

105

102

103

104

105

Pacific Blue

Alexa Fluor 488

Stain with Ab

Combine and stain with Ab

Cell number
102

103

104

105

102

103

104

105

Analyze phosphorylation

Deconvolute and analyze phosphorylation

Alexa Fluor 700

Alexa Fluor 750

Phospho-antibody fluorescence

Figure 1the fluorescent cell barcoding (FCB) technique. (A)Sampleonewasunstimulated,sampletwowasstimulated,andsamplethreewas treatedwithasmall-moleculeinhibitorbeforestimulation.Afterfixation,cellsinstandardphosphoflow(left)werepermeabilizedwithcoldmethanol, washed,andstainedwithphospho-specificantibodies.IntheFCStechnique(rightside),eachsamplewaspermeabilizedwith2025Cmethanol containingadifferentconcentrationofamine-reactivefluorescentdyes(FCBmarkers),yieldingauniquefluorescencesignatureforeachsample. Sampleswerethenwashed,combinedintoonetube,andstainedwithantibodies.Duringsoftwareanalysisoftheacquireddata,thesampleswere deconvolutedbacktotheoriginalsamplesbasedontheirFCBsignature.InbothstandardandFCBphosphoflowtechniques,fluorescenceofthe phospho-specificantibodyineachsamplewasmeasured.Intheplots,dottedlinesindicateautofluorescenceandredhistogramsrepresentsample fluorescence.(B)EfficientlabelingoffoursamplespermarkerwiththeFCBtechnique.U937cellswerefixed,thenpermeabilizedinmethanolcontaining 0,0.04,0.2,or1g/mlPacificBlueNHS,AlexaFluor488NHS,AlexaFluor700NHS,or0,0.4,2,or10g/mlAlexaFluor750NHSfor15minutesat 2025C.Afterwashingtwice,samplesstainedwitheachFCBmarkerwerecombinedandanalyzed.Shownarehistogramsidentifyingthefouroriginal samplesbarcodedwitheachFCBmarker.Graypeaksrepresentunlabeledsamples(zeroFCBmarker).Coloredpeaksrepresentsamplesreceivinglow, medium,andhighamountsoftheFCBmarker,withcolorintensitycorrelatingtoFCBmarkerstaininglevel.ReprintedbypermissionfromMacmillan Publishers,Ltd.:Nature Methods3:361368(2005).

2007InvitrogenCorporation.Allrightsreserved.TheseproductsmaybecoveredbyoneormoreLimitedUseLabelLicenses(seeInvitrogencatalogorwww.invitrogen.com).Byuseoftheseproducts youacceptthetermsandconditionsofallapplicableLimitedUseLabelLicenses.Forresearchuseonly.Notintendedforanyanimalorhumantherapeuticordiagnosticuse,unlessotherwisestated.

probes.invitrogen.com | 31

F lo w C y to m e t r y

Dead-cell discrimination in a different light

sytoX reD DeaD Cell staIN.
Cell count
200 Live 150 Dead 100

Thousandsofflowcytometryrunseverydayrelyondead-celldiscriminationeitherasan endpointresultorasoneparameterinabroaderexperimentalquery.Fluorescentmarkers fordead-cellstainingarethereforeindispensablereagentsforflowcytometricanalysis. Propidiumiodide(PI)haswidelybeenutilizedasastaintodifferentiateliveanddead cellsinflowcytometryexperiments.Becauseofitsbroademissionspectrum,multiple detectionchannelsontheflowcytometerareoccupiedwhenPIisused,whichlimits theemissionofPIdirectlyoverlapstheemissionofR-phycoerythrin(R-PE),makingPI incompatiblewithR-PElabeledprobes.Dead-cellstainsthatcanbeexcitedbylight sourcesotherthanthe488nmlaserarehighlysought-afterreagentsbecausethey allowthe488nmlasertobereservedforbrightfluorochromesonantibodiesdirected againstchallengingantigens.

50

0 82 0 102 103 104 105

SYTOX Red

105

CD8 R-PE

thenumberofparametersthatcanbedetectedinagivenexperiment.Inaddition,

104

103 0

1,656 5,894

103 0 103

104

105

CD4 R-PE Alexa Fluor 700

105

CD3 Alexa Fluor 488

SYTOXReddeadcellstainisahigh-affinitynucleicacidstainthateasilypenetrates cellswithcompromisedplasmamembranes,butwillnotcrossintactcellmembranes. AfterabriefincubationwithSYTOXRedstain(excitation/emission~640/658nm),the nucleicacidsofdeadcellsfluorescebrightredwhenexcitedwith633or635nmlaser light.AndbecauseSYTOXReddeadcellstaindoesnotrelyonthe488nmlaserand hasanemissionsignallimitedtoonechannel,addingittootherdyesinmulticolor lysedwholebloodsamplewithmouseantihumanCD4R-PEAlexaFluor700,mouse antihumanCD8R-PE,andmouseantihumanCD3AlexaFluor488for30minutes, followedby5MSYTOXRedstain,allowedeasygatingonthelive-cellpopulation andclearvisualizationofthevariousimmunophenotypes(Figure1).Theseproperties, combinedwithits>500-foldfluorescenceenhancementuponnucleicacidbinding, maketheSYTOXReddeadcellstainasimpleandquantitativesingle-stepdead-cell indicatorforusewithredlaserequippedflowcytometers. flowcytometryexperimentsiseasy.Forexample,staininganammoniumchloride

104

103

263 5,894 103 0 103 104 105

CD4 R-PE Alexa Fluor 700

105

CD3 Alexa Fluor 488

104

103

263 1,656

0 103

104

105

CD8 R-PE

Product
SYTOXReddeadcellstain*for633or635nmexcitation* *5MsolutioninDMSO*

Quantity
1ml

Cat. no.
S34859

Figure 1Illuminate your samples with better dead-cell discrimination. Thebloodcellsample wasstainedasdescribedinthetext.UsingaBD LSR II system (BD Biosciences), the sample was gatedonlymphocytesandoncellsnegativefor SYTOX Red stain. Fluorescence was monitored using 488 and 633 nm excitation and 530/30, 585/42,720/20,and660/20nmbandpassfilters.

32 | BioProbes52 | March2007

2007InvitrogenCorporation.Allrightsreserved.TheseproductsmaybecoveredbyoneormoreLimitedUseLabelLicenses(seeInvitrogencatalogorwww.invitrogen.com).Byuseoftheseproducts youacceptthetermsandconditionsofallapplicableLimitedUseLabelLicenses.Forresearchuseonly.Notintendedforanyanimalorhumantherapeuticordiagnosticuse,unlessotherwisestated.