[PDF] The Relevance of Flow Cytometry for Biochemical Analysis

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IUBMBLife,51:231-239,2001

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CriticalReview

TheRelevanceofFlowCytometryforBiochemical

Analysis

Jos´e-EnriqueO"Connor,1RobertC.Callaghan,2MartaEscudero,1Guadalupe Herrera,1AliciaMart´õnez,1Mar´õa-do-C´euMonteiro,3andHilarioMontol´õu1

1CentrodeCitometr´õa,DepartamentodeBioqu´õmicayBiolog´õaMolecular,FacultaddeMedicina,

UniversidaddeValencia,Valencia,Spain

2DepartamentodePatolog´õa,FacultaddeMedicina,UniversidaddeValencia,Valencia,Spain

3DepartamentodeBioqu´õmica,InstitutoSuperiordeCiˆenciasdaSa´ude-Norte,GˆandraPRD,Portugal

Summary

Flowcytometry(FCM)allowsthesimultaneousmeasurement ofmultiple?uorescencesandlightscatterinducedbyillumination ofsinglecellsormicroscopicparticlesinsuspension,asthey?ow rapidlythroughasensingarea.Insomesystems,individualcells orparticlesmaybesortedaccordingtothepropertiesexhibited. Byusingappropriate?uorescentmarkers,FCMisuniqueinthat multiplestructuralandfunctionalparameterscanbequanti?edsi- multaneouslyonasingle-particlebasis,whereasuptothousandsof biologicalparticlespersecondmaybeexamined.FCMisincreas- inglyusedforbasic,clinical,biotechnological,andenvironmental studiesofbiochemicalrelevance.Inthiscriticalreview,wesumma- rizethemainadvantagesandlimitationsofFCMforbiochemical studiesanddiscussbrie?ythemostrelevantparametersandana- lyticalstrategies.Graphicalexamplesofthebiologicalinformation providedbymultiparametricFCMarepresented.Also,thisreview containsspeci?csectionson?owcytoenzymology,FCManalysisof isolatedsubcellularorganelles,andcell-freeFCM.

IUBMBLife,51:231-239,2001

KeywordsCellfunction;cellsorting;?owcytometry;?uorescence; ?uorochromes;isolatedsubcellularelements;multiplexed assay.

INTRODUCTION

Flowcytometry(FCM)allowsthesimultaneousmeasure-

mentofmultiple?uorescencesandlightscatterinducedbyillu- minationofsinglecellsormicroscopicparticlesinsuspension,

Accepted24May2001.

AddresscorrespondencetoDr.Jos´e-EnriqueO"Connor,Centrode Citometr´õa,DepartamentodeBioqu´õmicayBiolog´õaMolecular,Facul- taddeMedicina,Avda.BlascoIb´a˜nez,17.46010-Valencia.Fax:+34-

963-864186.E-mail:jose.e.oconnor@uv.es

asthey?owrapidlythroughasensingarea(1,2).Insomesys- tems(cellsorters),individualcellsorparticlesmaybephysically separatedaccordingtotheirproperties(Fig.1).Thus,FCMis uniqueinthatmultiplebiologicalparameterscanbequanti?ed simultaneouslyonasingle-particlebasis,whileuptothousands ofeventspersecondmaybeexamined.Asaresult,largeand heterogeneouscellpopulationsaredescribedbasedonthebio- metricpropertiesoftheirindividuals(Table1). Becauseofitshistoricaldevelopment(1)anditsimportant clinicalimplications,thelargestbodyofcurrentapplicationsis diagnostic/prognostic,basedonimmunophenotypingandDNA contentassays(3,4).However,FCMisnowachoicemethodol- ogyinbasicandappliedstudies,includingcellularandmolec- ularbiology(5,6),biotechnology(7),toxicology(8),microbi- ology(9),plantphysiology(10),andoceanography/limnology (11).Ontheotherhand,clinicalFCMincreasinglyimplements biochemicalassaystoimprovesensitivityofabnormalcelliden- ti?cation(12)andtoprovidefunctionalinformationaboutpatho- geneticmechanismsinvolvedindiseaseconditions(13).

FCMAnalysisofCellBiochemistry:Parameters

andProbes Individualcells,bearingmultiplemarkersontheirsurface, containintracellularcompartmentswiththeirownmetabolic environment.Functionalintegrityofmembranesisnecessary fortheregulationofsuchcompartmentsthat,inturn,condition metabolicpathwayswithinthem.Inmanycases,homeostasisof cellcompartmentsrequiresregulatedtransportacrosscellmem- branes.Ontheotherhand,plasmamembraneisdeeplyinvolved inbiochemicalresponsesthatmediatecellactivationtriggered bymultiplestimuli.Manyoftheseresponsesaredependenton speci?creceptorsandtriggerconsistentchangesinionicstatus andenzymeactivitiesinsignaltransductionpathways,leading 231

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Figure1.Schematicsofatypical?owcytometry/cellsortinginstrument.Theschemeshowshowcellsareforcedtocrossalaserbeamatthe?owchamber.Multiple?uorescenceandlightscattersignalsarecollectedanddirectedthroughthe?uorescencecollectinglensand?lterassembly.Separatephotomultipliersorphotodiodesamplifythesignalsaccordinglyandtheinstrumentelectronicsprocessesandclassi?esthepulsesgeneratedbysinglecellsorparticles.Insomesystems(Cellsorters)cellsmaybeseparatedbyelectrostaticchargingdevicesafterbreakingthe?owintomicroscopicindividualdropletscontainingsinglecells.Theinterfacedcomputerreceivestheprocessedsignalsinrealtimeandallowsthecontrolofinstrumentalsettingsforanalysis(cytosettings)andcellsorting(sortsettings),aswellasprede?nitionoftheprotocolformultiparameterdataacquisition.TheresultsofanalysisareformattedasFCS?les(FlowCytometryStandard)andmaybedisplayedasdifferentgraphicrepresentationsofcellpopulationdistribution(univariatehistogramsormultivariatedotplots)whereuser-de?nednumericalandstatisticalanalysisisperformedbydedicatedsoftware.Datamaybestoredasnoncorrelateddatamatrixforeachsinglecell(listmode?les)inawaypermittingtoreproducevirtuallyandinstantlythedataacquisitionprocess,whilekeepingorchangingasdesiredsomefeaturesoftheanalysis,suchasgatede?nition,parametercorrelation,typeofdisplay,andsoon.FCS?lesmaybesharedandtransferedbyphysicalsupportsystems(?oppydisks,CDdisks)orbylocalorglobalnetworkstootherindependentcomputerstoproceedtofurtherdataanalysis,includingtheuseofthird-partysoftwareforFCMapplications.

FLOWCYTOMETRYINBIOCHEMICALANALYSIS233

Table1

Biochemicalassaysby?owcytometry:samples

andprobes

Typeofbiologicalsamples

²Pluricellularorganisms²Isolatednuclei

²Cellspheroids²Subcellularelements

²Hybridomas²Chromosomes

²Cellfusions²Liposomes

²Humancells²Mallorybodies

²Animalcells²Amyloidplaque?bers

²Plantprotoplasts²Membranefractions

²Prokaryoticcells²Viralparticles

²Yeasts²Solubleantigens1

²Microalgae²DNAsequences1

Typeof?uorochromesand?uorescentmarkers

²Fluorochromesreacting²FluorescentpHindicators withspeci?cchemical²Fluorescentionchelators groups²Membrane-potential

²Fluorochromepairsforsensitivedistribution

resonanceenergytransfer?uorescentdyes ²Fluorescentantibodies²Fluorogenicsubstratesof

²Fluorescentlectinsintracellularenzymes

²Fluorescentnucleicacid²Fluorescentmacromolecules sequences²Fluorescentsynthetic

²Fluorescentlipidsparticles

²Endogenous?uorescent

molecules

1Using?uorescentmicrospheresascapturereagentsand?uores-

centligandsasreportermolecules. ultimatelytoregulationofgeneexpression,celldiferentiation, and/orproliferation.

FCMisappliedsuccessfullytostudyeachstepofthisvast

complexityofcellularbiochemistry.Formostapplications,cells mustbestainedwith?uorescentmarkersofde?nedopticaland biologicalproperties(Table1),butFCMtakesalsoadvantage ofendogenous?uorochromesrelatedtointracellularfunctions (1).Inthisway,assummarizedinTable2,therangeofparam- etersavailablefortheFCMevaluationofcellbiochemistryhas beenextendedfrombroadassessmentofcellbehaviourtoquan- ti?cationofsinglemoleculesundergoingorregulatingspeci?c biochemicalreactions. Asigni?cantpartofFCMstudiesinvolvesanalysisofthese parametersinrelationtocellactivation(14,15)andprolifera- tion(16),cellsensitivity(17,18)orresistancetodrugaction (19),andcelldeathbyapoptosisornecrosisinawiderangeof experimentalsettings(20-22).Althoughmostofthesestudies fallwithinthescopeofbasicresearch,thedevelopmentofsim- pleassaysfortheseparametershasallowedtheirapplicationto differentclinicalsituations(3,23).

Speci?cFeatures,StrategiesandLimitations

ofFunctionalFCM

BecauseoftheuniquefeatureofFCM,i.e.,themultipara-

metricexamination(andphysicalseparation)ofsinglecellsor particlesatveryfastrate,thisparticulartechniqueofbiochem- icalanalysishasevidentadvantagesoverotherconventional methodologies.Thus,thelargenumberofcellsanalyzedand theinstrumentalsettingsofcurrentcytometersprovidemulti- plestrategiestoobtainprimaryinformation,andallowalarge numberofgeneralapplications,asTable3attemptstocover.

Fromapracticalpointofview,themainassetsofFCMcanbe

summarizedasfollows: MultiparametricDataAcquisition.Moststandardbiochem- icalproceduresdetermineasingleparameterperassayandare notsensitiveenoughforsingle-cellanalysis.FCMinstruments allowroutinelytwomorphology-relatedparameters(forward- andside-lightscatter)and3-5?uorescencesignalspersingle particle.Inthisway,inasingle-tubeassay,oneormoreparam- etersmaybeusedtoidentifyandselect("gatedanalysis")cell subsetsinheterogeneouspopulations(e.g.,live,apoptotic,or necroticcells;cellsofdifferentoriginorlineage;cellsindif- ferentcellcyclestageandsoon),whereasothersignalsmay beassignedtoanalyzespeci?cstructuresorfunctionsinthese selectedpopulations.Anexampleofthisconceptisillustrated inthesingle-tubeassayshowninFig.2.Thenumberofavail- ableparameterspersinglecellincreaseswhenmultiple-lasercy- tometersareused.Obviously,theanalysisofmultiplealiquots persampleallowstoexpandinde?nitelythenumberofparam- etersbycombiningseparately?uorescentmarkersofdifferent biologicalpropertiesbutsimilaropticalproperties.Anexample ofthisconceptisillustratedintheintegratedanalysisshown inFig.3.ThistypeofFCManalysis(panelanalysis)isthe hallmarkofimmunohematology,wheretypicallymorethan20 ?uorescentmonoclonalantibodiesagainstepitopesinleukocyte plasmamembranemaybeusedfortypingleukemiasandlym- phomas(4,24).

MultivariateDataAnalysis.Duetothehardwareandsoft-

waredesignofmoderncytometers,multiparametricacquisition isinterfacedtomultivariatedataanalysis.Inthisway,acell populationisnotdescribedbymereenumerationoftheindivid- ualpropertiesmeasuredbutbytheircorrelationonasingle-cell basis,thusincreasingthediscriminatingpower.Moreover,the possibilityofstoringFCMdataasanuncorrelateddatamatrixfor eachanalyzedcell("listmode?les")allowsonetode?ne,ifnec- essary,newparametriccorrelationsandpopulationselectionby replaying(off-line)thoseelectronic?les.Thisisaninvaluable toolespeciallywhensmallorinfrequentsamplesarestudied.

FastAnalysisofLargeNumberofLiveCells.FCMmaybe

performedonalargevarietyofbiologicalmaterialindifferent conditionsofvitality(e.g.,intactfreshcells,?xedand/orper- meabilizedcells),asindicatedinTable3.Theuseoflivecells allowsonetoprobemultiplebiochemicalparametersinmini- mallyperturbedintracellularenvironments,aswellasinnear- physiologicalextracellularconditions.

234O"CONNORETAL.

Table2

Biochemicalassaysby?owcytometry:parameters

CellsurfaceparametersCytosolicparametersNuclearparametersSubcellularelements MembraneintegrityGeneralproteinDNAcontentNormalmitochondria MembranepotentialMitochondrialactivityRNAcontentMegamitochondria MembranerecyclingMitochondriacontentNucleartotalproteinsCis-Golgivesicles ReceptorexpressionCytosolicpHNuclearspeci?cproteinsTrans-Golgivesicles ReceptorinteractionsLysosomalpHChromatinconformationEndosomes ReceptormodulationTyrosinephosphorylationCyclinsandCDksPhagosomes SurfaceglycoconjugatesCytosolicCa2CProliferation-relatedantigensChloroplasts LigandbindingtosurfacereceptorsROSandNOSDNAsynthesisThylakoids Cell-celladhesionEnzymeactivity:DNAstrandbreaksExtracellularanalytes

Membrane?uidityOxidasesDNAoxidation

CholesterolcontentDehydrogenasesDNArepair

LossoflipidassimetryEsterasesNuclearreceptors

Permeabilityto?uorescentprobesProteasesGeneexpression

MembraneperoxidationTransferasesGenereporting

MembranesheddingProteinmodi?cation

EndocytosisFreesolublethiols

PhagocytosisGlutathione

PynocytosisProteinthiols

Ef?uxpumpsNonpolarlipids

BacterialcellwallPolarlipids

YeastcellwallCytoskeletalproteins

Granulecontent

Thefastrateofdataacquisitionandthepossibilityofex- aminingmillionsofindividualparticlesinareasonabletime allowsthedetectionandaccurateanalysisofinfrequentorrare cells,downto1eventper108cells(25).Suchapossibilityisin contrastwithbulkstandard?uorimetricdeterminationsinwhich millionsofcells(ortheirextracts)areanalyzedatthesametime, yieldingasinglemeanconcentrationvalue.

IndividualCellSorting.SomeFCMsystemsareableto

separatephysicallyindividualcellorparticlesaccordingtotheir cytometricproperties.Themostadvancedcellsortersarebased onelectromagneticde?ectionofindividualdropletsgenerated byhigh-frequencyvibrationofthe?owchamber(1).Insuch systems,uptofourdifferentsubpopulationscanbesortedsimul- taneouslyor,ontheotherhand,onesinglecellcanbedeposited inagivenpositionofamicrowellarray.Cellsortingallowsthe combinationoftheintrinsiccapabilitiesofFCMresultswith informationobtainedbyimage(conventionalandconfocalmi- croscopy)andmolecular(polymerasechainreaction,insituhy- bridization)techniques,andprovidesapreparativetoolforrapid isolationoflivingrarecellsofbiochemicalrelevance,suchas stemcells(26),transfectants(27),orhybridomasproducinga givenantibody(1).Itisworthmentioningthecontributionof ?owsortingofchromosomestothesequencingofthehuman genome(28,29). AsindicatedinTable4,therearealsocriticalpointsanddif?- cultieswhenperformingadequatefunctionalanalysisbyFCM, whichmostlydependonthemaintenanceofadequateviability ormetaboliccapacityofcellsandsubcellularelementsaswell asavoidingtheinterferenceof?uorescentprobeswithcellular functions.

FCMApproachtoClassicBiochemistry:

FlowCytoenzymology

Currently,awiderangeof?uorescentsubstratesortheir?uo- rogenicprecursorsareavailableforFCManalysis(1,2,30).On theotherhand,?owcytometersincorporatetimeasaparameter tofollowthekineticsof?uorescencevariationsonthespeci?c modi?cationofsubstrates(14,31).Forthesereasons,?owcy- toenzymology(30)appearsasapromisingapplicationofFCM foranalysisofmetabolism.Flowcytoenzymologyisappliedto agrowingnumberofenzymaticactivitiesinmultiplebiochem- icalpathways(32-34)andthesestudiesmayhaveadirectclin- icalimpact(35,36).Thus,theyarecurrentlyappliedtoassess leukocytefunction,tocorrelatecellmetabolismandmalignant capacityinfreshtumorcells,andtoevaluatedrugmetabolism andtherapymonitorizationinpharmacologicalstudies(37).

FlowCytometricAnalysisofIsolated

SubcellularCompartments

Theuseof?owcytometerstoanalyzefunctionalpropertiesof isolatedsubcellularparticlesislessfrequentthanitsapplication

FLOWCYTOMETRYINBIOCHEMICALANALYSIS235

Table3

Biochemicalassaysby?owcytometry:strategies,information,andapplications AssaystrategiesPrimaryinformationMaingeneralapplications a)Accordingtothebiological material:

²Assaysusingfreshcells

²Assaysusing?xedcells

²Assaysusingsubcellularelements

²Multiplexedassays

b)Accordingtospeci?ccellselection:

²Nongatedassays

²Gatedassays

c)Accordingtoassayduration:

²Singleend-pointassays

²Sequentialend-pointassays

²Kineticassayswithunperturbed

cells

²Kineticassaysfollowingcell

stimulationwithligands d)Accordingtodataanalysis:

²On-lineanalysis(realtime)

²Off-line(Listmodeanalysis)

²Intensityofexpressionofmultiple²Identi?cation/characterizationof parameterswithinhomogeneouscellsbaseduponmultiple cellpopulationsbiochemicalparameters ²Heterogeneityofexpressionofmultiple²Diagnosticapplications,including parametersincellsubpopulationsdetectionofrarepathologicalcells ²Correlationbetweendifferentparameters²Analysisofcellactivation, incellpopulationsincludingreceptorbiologyand ²Ratiobetweenmultipleparameterssignaltransduction insinglecells²Analysisofgeneexpression, ²Evolutionoffastand/ortransientincludinggeneengineering dynamicparameters²Analysisofcellcycleand ²Evolutionofslowand/orsustainedproliferation-relatedevents dynamicparameters²Analysisofdifferentiation ²Detectionandanalysisofrare²Flowcytoenzymology cells/particles²Analysisofcellviability ²Correlationwithparametersanalyzedandcelldeath,including withothertechniquesfollowingapoptosisandnecrosis cellsorting²Analysisofmicrobialbiochemistry, includingsensitivitytodrugs

²Controlofbiotechnological

processes,includinggrowth conditionsandproductivity

²Environmentalbiochemistry

inwholecellstudies.However,mostcurrentinstrumentsare adequatelysensitiveforsubcellularanalyses,whichhavealways beenahallmarkofbiochemistry. Somemethodologicalaspectsbecomecriticalwhenanalyz- ingsinglesubcellularparticlesbyFCMbecauseoftheirsmall size,thedifferentpermeabilityoruptakerateofdyesbyiso- latedorganelles,andtheirusuallyincreasedlability.However, FCManalysisofisolatedorganellesprovidesinsightofsubcel-

Table4

Biochemicalassaysby?owcytometry:dif?culties

Criticalpoints

²Preparationofsingle-cellsuspensionsfromadherentcell populations ²Maintenanceofcellviabilityalongtheexperimentalperiod ²Isolationofsubcellularelementsfromcellsandtissues

²Readjustingconditionsforsubcellularanalysis

²Identi?cationofsmallcellsandparticlesfrombackground noise ²Adequateaccessofprobestointracellularsitesorprocesses

²Adequateretentionofsubstratesandprobes

²Noninterferenceofprobeswithcellfunctions

²Adequateselectionoftime-windowsforkineticassays ²Assaycalibrationfordataexpressioninabsoluteunits lularfunctionsandstructuresinexperimentalmodelswherea higherdegreeofmetaboliccontrolcanbeachieved(Table1and

Table2).

Rhodamine123andothermembrane-potential(MP)-sensitive dyes(15,31,38)havebeenusedforfunctionalanalysisofiso- latedmitochondria,whereasotherMP-independentmito- chondrialdyescanbeappliedtodeterminethemitochondrial contentinwholecells(39).Manipulationofmembranepotential inisolatedmitochondriainducedpredictablechangesinRh123 ?uorescenceandrevealedmitochondrialheterogeneityinliver cellsandheterogeneousresponsestophysiologicaland nutritionalconditions(40,41).Isolatedmitochondriahavebeen usedalsofortoxicologicalandpharmacologicalstudies,which yieldeddatacomplementarytothoseobtainedusingwholecells (42).

FCMhasbeenappliedalsotoanalyzethebindingof?uo-

rescentlectinstoisolatedchloroplasts(43)andGolgivesicles (44)forthestudyoftheiroligosaccharidecontent.Thedatathus obtainedmaybeofrelevancetothestudyofthenormaland alteredmechanismsofglycoproteinmaturationandsorting.

Cell-FreeCytometry:QuantifyingSolubleAnalytes

FCMisnotlimitedtotheanalysisofbiochemicalcompo-

nentsinsuspensionsofcellularorsubcellularparticles.Onthe contrary,arecentlydevelopedstrategyknownasmultiplexed

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Figure2.Exampleofthemultiparametricdataacquisitionandmultivariatedataanalysisinasingle-tubeFCMbiochemicalassay.First,25ⁱL-samplesofhumanwholebloodwereincubatedwith5ⁱLofpan-leukocyteantibodyCD45-PCy5for15minanddilutedto1mLwithRPMImedium.Then,5ⁱLofpH-indicator,1mMBCECF-AM,wereaddedandthesampleincubatedfor15minat37±Cinthedarkinthepresenceorabsenceof4ⁱMethyl-isopropyl-amyloride(EIPA),aNaC/HCantiportinhibitor.SampleswererunonanEPICSXL-MCL?owcytometerandthefollowingparametersacquired:Forwardscatter(FS,anestimationofcellsize),sidescatter(SS,anestimationofcellgranularity),LogFL1(BCECF-AMgreen?uorescence),Log-FL2(BCECF-AMyellow?uorescence),LogFL4(CD45-PCy5red?uorescence),RatioFL2/FL1(theratiobetweenBCECF-AMyellowandgreen?uorescences),anestimationofintracellularpH(pHi),andTime(thex-axisscalerepresents300s).Samplewasrunfor10s,thenpaused,and50ⁱLof100mMpropionicacid(ProH)added(arrow).Dataacquisitionwasre-startedtoshowinducedacidi?cationasadecreaseintheratioFL2/FL1.Analysisofwholeblooddoesnotdistinguishleukocytes(WBC)fromerythrocytes(RBC)basedinscatterparameters(A).However,gatingonCD45showsclearlyleukocytesubpopulations(B)andtheirtypicalmorphology(C).AnalysisofBCECFFL1andFL2separately(D)doesnotprovideinformation.However,bivariateplotofFL2/FL1ratiovsSS(E)showsthatleukocytesubpopulationsdifferinrestingpHi,granulocytesbeingmorealkaline.KineticanalysisofpHifollowingacidi?cationwithProHshowsheterogeneityinWBC(F).Selectionofspeci?csubpopulationsondotplot(C)showsthatlymphocytesdonotrecoverpHi(G),whereasmonocytes(H)andgranulocyte(I)returnrapidlytorestingpHi.TheparticipationofNaC/HCantiportiscon?rmedbytheinhibitoryeffectofEIPAinasecondtuberuninthesameconditions(J).

FLOWCYTOMETRYINBIOCHEMICALANALYSIS237

Figure3.Exampleofanintegrated(multipletubes)?owcytometricassayofbiochemicalparameters:Studyofplateletactivationinwholeblood.(A)Plateletsareidenti?edinwholebloodwithanantibodyspeci?cforglycoproteincomplexgpIIb-IIIa(CD41-PE).(B)RiseincytoplasmicCaCCdetectedbythegreen?uorescentchelatorFluo-3AMfollowingplateletactivationwithADP.(C)Rapiddegranulationresponseuponplateletactivationfollowedbykineticanalysisoflossofthecomplexity-dependentsignalSS.(D)Rearrangementofcytoskeleton(changeofactinGtoactinF)usingFITC-labelledphalloidininplatelets?xedfollowingadditionofADP.ThebiochemicaleventsdepictedinpanelsB-Dareconsideredveryearlyeventsinthefunctionalchangesinducedinplateletbyactivatingagonists.ThesurfaceexpressionofphosphatidylserinecanbeanalyzedkineticallywithFITC-labelledannexinVfollowingadditionofcalciumionophoreA23187,anexperimentalmodelofinductionofplateletpro-coagulantsurface(E).Undertheseexperimentalconditions,plateletsreleasemembranemicroparticles,asevidencedbythelossofconstitutivemembraneglycoproteinCD41(F).DatainthisgraphareobtainedbydisplayingCD41?uorescenceinannexinV-positivecellsatthelastofthetimeslicesde?nedintheplotofpanelD.Theoverexpressiononplateletsurfaceofthe®-granuleproteingmp140(CD62-P)isoneofthelatestsequentialbiochemicalchangesfollowingpseudophysiologicalplateletactivationwithADP(G).

238O"CONNORETAL.

analysisallowssimultaneousquanti?cationofmultipleanalytes insolution.Thebasisforeachmeasurementconsistsofasetof microspheresidenti?ablebyembedded?uorophores.Individual setsofmicrospheresaremodi?edwithreactivecomponentssuch asantigens,antibodies,oroligonucleotides,andthenmixedto allowmultipleindependentreactionstobeanalyzedsimultane- ously.Theuseofmicrosphereswithdifferentratiosofredand orange?uorescenceprovidesthemultiplexedformat,andFCM analysissimultaneouslyidenti?esboththemicrospheretypeand the?uorescentgreensignal,revealingthecaptureofthepartic- ularanalyte(45).Thismeasurementsystemcananalyzeupto

64analytesinasinglesample.

Withthemicrosphere-associatedtechnology,theapplica- tionsforbasicandclinical?owcytometryinthefutureare enormous.Forinstance,thesystemhasbeenusedtoperform simultaneousdetectionofmultiplex-ampli?edhumanimmu- node?ciencyvirustype1RNA,hepatitisCvirusRNA,and hepatitisBvirusDNA(46).Thisapproachhasbeenfound tobemoreaccurate,sensitive,andreproduciblethanthecon- ventionalmicrotitreELISAforqualitativeandquantitative immunoassaysforseveralproteins.Forinstance,thisassaycan accuratelyquantitate15cytokinesin100ⁱL-samples,whereas thesameanalysisbyELISArequires1.5mL(100ⁱLfor eachcytokineassay)(47).Also,multiplexed?owcytometric analyseshavebeendevelopedtomeasuresimultaneously cytokinereceptorexpression,internalcytokineexpression, andcytokinesecretionbyactivatedT-cellsinvitro(48),thus openinganinterestingapproachtothestudyofcellactivation responses. Aseriesofnovelapplicationsillustratesthepotentialofge- nomicanalysiswithmicrospherearraysandFCMusingsub- nanomolarconcentrationsofsampleinsmallvolumesatrates ofonesampleperminuteorfaster,withoutawashstep.Thus,the systemhasbeenusedtoperformDNAsequenceanalysisbymul- tiplexedcompetitivehybridizationofsequence-speci?coligonu- cleotideprobes(49)andformultiplexedanalysisofdozensof singlenucleotidepolimor?sms(50).Theseresultsdemonstrate thesensitivityandaccuracyof?owcytometry-basedminise- quencing,apowerfulnewtoolforgenome-andglobal-scale

SNPanalysis.

Thereisasigni?cantnumberofwebsitesdedicatedtoba-

sicFCMthatshouldbevisitedtoobtainfurtherinformationas wellasrelatedlinksandFCMfreeware.Forthesakeofbrevity, readersareencouragedtobookmarkhttp://www.biochem.mpg. de/valet/cytorel.html(Cytorelay,Max-PlanckInstitutforBio- chemistry);http://?owcyt.cyto.purdue.edu/(PurdueUniversity CytometryLaboratories),andhttp://www.bio.umass.edu/ mcbfacs/?owhome.html(FlowCytometryFacility,University ofMassachussetsatAmherst).

ACKNOWLEDGMENTS

TheresultsofFigs.2and3areunpublisheddatafromresearch projectssponsoredbytheEuropeanCommission(15348-1999-

10FIEDISPES),theGeneralitatValenciana(GV-D-VS-22-148-

96),PRODEP,andIzasa,S.A.

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