WB杂带的问题,出现两条带,还有就是只有一条带,但与对照组不在一条线...相关交流-蚂蚁淘在线

HerewepresenttheabsorptionandemissionspectraoftheFluorochromesBDBiosciencesPharmingenconjugatestomonoclonalantibodiesandotherproteins.Fourofthesefluorochromes,fluoresceinisothiocyanate(FITC),R-phycoerythrin(R-PE),BDCy-Chrome™,andPerCP,canbeusedwithsingle-laserflowcytometersequippedwithanargon-ionlaseremittinglightat488nmforthree-colorflowcytometricanalysis(Fig.1).

BDBiosciencesPharmingenalsooffersmonoclonalantibodiesandproteinsconjugatedtoallophycocyanin(APC)andavidinconjugatedtoTexasRed™.Bothofthesedyesareusefulinexperimentswheremulti-coloranalysisisdesiredusingflowcytometerswithduallasercapABIlities(Fig1).APCcanbeexcitedbyahelium-neon(HeNe)laseremittinglightat633nm,byakryptonlaseremittinglightat647nm,orbyadyelaserwhichcanbeconvenientlytunedtoemitlightinthe550-650nmrange.(Inadyelaser,thelasingmediumisasolutionoffluorescentdyeexcitedbyapumplaser,usuallyanionlaser.)TexasRed™,availableconjugatedtoavidin,canbeexcitedbyanargon-kryptonmixed-gaslaserat568nm,orwithadyelaser,wherebothAPCandTexasRed™canbeusedsimultaneously.Becausemanycombinationsoflasers,detectors,filtersandfluorochromesarepossIBLeformulti-coloranalysis,properprecautionsneedtobetaken(i.e.,bandpassfilters,dichroicmirrors,longpassfilters,etc.)bytheoperatortoensureeachfluorochromeisbeingdetectedbyonlyonedetector(Fig.1).Inthefollowingdescriptions,wegiveourrecommendationsfortheidealinstrumentset-upforusewithourreagents.(summarizedinTable1)

Allophycocyanin(APC)isanaccessoryphotosyntheticpigmentfoundinbluegreenalgae.Itsmolecularweightisapproximately105kDa.APChas6phycocyanobilinchromophorespermolecule,whicharesimilarinstructuretophycoerythrobilin,thechromophoreinR-PE.Ithasa650-nmwavelengthabsorptionmaximum(Fig.2)anda660-nmfluorescenceemissionmaximum(Fig.2).Usinga660?10nmBPfilterwillgiveoptimumdetectionforthisfluorochrome.APCcanbeusedinflowcytometersequippedwithduallasersformulti-coloranalysis(Fig.2).Itcanbeexcitedbylaserlightbetween600-640nm.Forthis,werecommendaHe-Nelaserat633nm,oratunabledyelasertundbetween600-640nm.

BDCy-Chrome™isatandemconjugatesystem,withanabsorptionmaximumofapproximately650nm(Fig.2),whichcombinesR-phycoerythrinandacyaninedye(MW1.5kDa).Whenexcitedby488-nmlight,theexcitedfluorochrome(R-PE)isabletotransferitsfluorescentenergytothecyaninemolecule,whichthenfluorescesatalongerwavelength.Theresultingfluorescentmaximumisapproximately670nm(Fig.2).Usinga650-nmlongpassfilterwillgiveoptimumdetectionforthisfluorochrome.TheefficiencyofthelightenergytransferbetweenthetwofluorochromescanbeseeninFig.2Fwherelessthan5%oftheabsorbedlightislostasfluorescenceat575nmbyR-PE.Comparedtootherfluorescenceenergy-transfersystemsusedinflowcytometry(e.g.,RED613™,EDC,PerCP),BDCy-Chrome™isasuperiorfluorochromeforthirdcoloranalysisbecauseofitshighemissionintensityandbroadspectrum.AswithourR-PEconjugates,anaverageofoneBDCy-Chrome™moleculeiscoupledperantibodyorprotein.Becauseofitsbroadabsorptionrange(Fig.2),BDCy-Chrome™isnotrecommendedforusewithdual-laserflowcytometerswhereexcitationbybothlasersispossible.

Fluoresceinisothiocyanate(FITC)isafluorochromewithamolecularweightof389daltonsandanabsorptionmaximumat495nm(Fig.2).Itsexcitationby488-nmlightleadstoafluorescenceemissionmaximumaround520nm(Fig.2).Usinga530?15nmbandpass(BP)filterwillgiveoptimumdetectionforthisfluorochrome.Theisothiocyanatederivative(FITC)isthemostwidelyusedformforconjugationtoantibodiesandproteins,butotherderivativesareavailable.FITChasahighquantumyield(efficiencyofenergytransferfromabsorptiontoemissionfluorescence)andapproximatelyhalfoftheabsorbedphotonsareemittedasfluorescentlight.ThenumberofFITCmoleculesperconjugatepartner(antibody,Avidin,Streptavidin,etc.)isusuallyintherangeofthreetofivemolecules.

R-phycoerythrin(R-PE)isanaccessoryphotosyntheticpigmentfoundinredalgae.Invivo,itfunctionstotransferlightenergytochlorophyllduringphotosynthesis.Invitro,itisa240-kDaproteinwith34phycoerythrobilinfluorochromespermolecule.ThelargenumberoffluorochromesperPEmoleculemakeR-phycoerythrinanidealpigmentforflowcytometryapplications.Itsabsorptionmaximumis564nm(Fig.2).Whenexcitedby488-nmlight,itsfluorescenceemissionmaximumisapproximately575nm(Fig.2).Forsingle-laserflowcytometeruse,werecommendusinga585?21nmBPfilterforoptimaldetection(Fig.1).Whenperformingmulti-coloranalysiswithadual-lasersystem,atighterwindowofdetectionisrequiredtocompensatefortheotherconjugatesbeingused(e.g.,TexasRed™).Forthis,werecommendusinga575?13-nmBPfilter(Fig.1).OurconjugationchemistryyieldsanaverageofoneR-PEmoleculeperantibodyorprotein.Theemittedlightiscollectedinthefluorescence-2(FL2)channel.

PE-TexasRed™isatandemconjugatesystemwhichcombinesR-PEandTexasRed™andhasanabsorptionmaximumofapproximately564nm.Whenexcitedby488-nmlight,theexcitedfluorochrome(PE)isabletotransferitsfluorescentenergytotheTexasRed™molecule,whichthenfluorescesatalongerwavelength.Theresultingfluorescentemissionmaximumisapproximately615nm.SpecialcaremustbetakenwhenusingPE-TexasRed™conjugatesinconjunctionwithR-PEasthereisconsiderablespectraloverlapintheemissionprofilesofbothfluorochromes.

Peridininchlorophyllprotein(PerCP)isacomponentofthephotosyntheticapparatusfoundinthedinoflagellate,Glenodinium.PerCPisaproteincomplexwithamolecularweightofapproximately35kDa.Whenexcitedbylightat488nmfromanargon-ionlaser,PerCPhasaexcitationmaximumaround490nm,withanemissionspectrumwhichpeaksat675nm.Theemittedlightiscollectedinthefluorescence-3(FL3)channel.Duetoitsphotobleachingcharacteristics,PerCPconjugatesarenotrecommendedforuseonstream-in-airflowcytometers.

PerCP-Cy5.5isatandemconjugatesystemthancombinesPerCPwithacyaninedye(Cy5.5™)andhasanabsorptionmaximumofapproximately490nm.Whenexcitedby488-nmlight,theexcitedfluorochrome(PerCP)isabletotransferitsfluorescentenergytothecyaninemolecule,whichthenfluorescesatalongerwavelength.Theresultingfluorescentemissionmaximumisapproximately694nm.Usinga650nmlongpassfilterwillgiveoptimumdetectionforthisfluorochrome.Theemittedlightiscollectedinthefluorescence-3(FL3)channel.PerCP-Cy5.5isrecommendedforusewithstream-in-airflowcytometers.

APC-Cy7isatandemconjugatesystemthatcombinesAPCandacyaninedye(Cy7™)andhasanabsorptionmaximumofapproximately650nm.WhenexcitedbylightfromadyeorHeNelaser,theexcitedfluorochrome(APC)isabletotransferitsfluorescentenergytothecyaninemolecule,whichthenfluorescesatalongerwavelength.Theresultingfluorescentemissionmaximumisapproximately767nm.Itisrecommendedthata750-nmlongpassfilterbeusedalongwithared-sensitivedetectorsuchastheHammatsuR3896PMTforthisfluorochrome.SpecialfiltersarerequiredwhenusingAPC-Cy7?inconjunctionwithAPC.ItisrecommendedthatspecialprecautionsbetakenwithPharRedconjugates,andcellsstainedwiththem,toprotectthefluorochromefromlong-termexposuretovisiblelight.

TexasRed™isasulfonylchloridederivativeofsulforhodamine101withamolecularweightof625daltons.BDBiosciencesPharmingenoffersTexasRed™,conjugatedtoavidin,asausefulsecondstepformulti-coloranalysis.Becauseitemitsinthelongwavelengthsofthedeepredregion(Fig2),TexasRed™haslittlespectraloverlapwithFITC.Whenperformingmulti-coloranalysisinvolvingbothTexasRed™andR-PE,BDBiosciencesPharmingenrecommendsexcitationofTexasRed™usingadual-laserflowcytometerequippedwithatunabledyelasertoavoid"leaking"intothePEdetector.Ifakryptonlaser,emittinglightat568nm,isused,thelaserlightwill"leak"intotheR-PEchannel.TexasRed™canbeusedinconjunctionwithAPCformulti-coloranalysiswhenbothdyesareexcitedinthe595-605nmrangewithadyelaser.TexasRed™hasanabsorptionmaximumof596nm(Fig.2).Itsemissionmaximum,whenexcitedby595-600-nmlaserlight,is615nm(Fig.2).Usinga620?10-nmbandpassfilterwillgiveoptimumdetectionforthisfluorochrome(Fig.1).

Comparativestainingusingamonoclonalantibody(RA3-6B2;anti-B220;Cat.No.557390/553084**)conjugatedtodifferentfluorochromesandanalyzedoneitherBDFACSVantage™(upperpanels)orBDFACSCalibur™(lowerpanels).Thenumbersindicatetheratioofthemedianfluorescenceintensityofpositivecellstothenegativecells(signaltonoiseratio).TheseplotsdemonstratehowchoicesinA)fluorochrome-conjugatesorB)instrumentationcanaffectthefluorescenceintensityobservedforagivenpopulation.

Thedifferencesobservedbetweenindividualfluorochrome-conjugatescanbeaffectedbythemAbconjugated.ThuswhileintheexampleabovethePE-conjugateisbrighterthantheBDCy-Chrome™-conjugate,whenanalyzedontheBDFACSCalibur™,formanymAbstheCy-Chrome™-conjugateresultsinabrighterstain.ContactBDBiosciencesPharmingenTechnicalServicesformoreinformationonspecificreagents.
Similarlydifferentflowcytometersutilizedifferentlasersanddifferentfluorescencefiltersetswhichcanresultindifferencesinsignaltonoiseratioswhenusingthesamereagent.NotethatPEreagentstendtobebrighterwhenusedonaBDFACSCalibur™whileAPCreagentsarebrighteronaBDFACSVantage™.Notechangesofthesignaltonoiseratiodependingonfluorchromeandinstrumentused.

Enlargeimage

Figure1.

"Topschematic."Asinglelaserflowcytometerwithfiveparametersofdetection.Twodetectorsdetectthelightscatter,andthreephoto-multipliertubes(PMTs)detectthefluorescentsignals.ThebandpassfiltersaresetupforoptimaldetectionwithBDBiosciencesPharmingen"sfluorochromes:FITC,PE,BDCy-Chrome™andBectonDickinson"sPerCP.

"Bottomschematic."Aduallaserflowcytometerwithsixparametersofdetection.Twodetectorsdetectthelightscatter,andfourPMTsdetectthefluorescentsignals.ThebandpassfiltersaresetupforoptimaldetectionwithBDBiosciencesPharmingen"sfluorochromesFITC,PE,APC,andTexasRed™.Thesecond(orange)laserlightisemittedfromatunabledyeheadusingrhodamine6Gasthefluorescentdyeforexcitation.Forwardlightscatter(FSC),sidescatter(SSC),FITC,andPEsignalsareallproducedbytheprimary488-nmargon-ionlaser.APCandTexasRed™signalsareproducedbythesecondlaser(dyeheadwitha488-nmargon-ionlaser).

Figure2.AbsorptionspectraofFluorochromes.Individualfluorochromeexcitationspectraarefoundingrayandthecorrespondingemissionspectrainblack.TypicalbandpassfiltersaregivenforeachfluorochromeasusedonaFACSVantage™exceptforBDCy-Chrome™andPerCPwhichareshownforFACSCalibur™configurations.

TABLE1.Comparisonofindividualfluorochromeswithsingleandduallaserflowcytometry.

Fluorochrome	LaserExcitationWavelength(nm)	FACScan™FACSCalibur™(1laser)	FACSCalibur™(2lasers)	FACSTar™FACStarPlus™FACSVantage™(1laser)	FACSVantage™SEFACStarPlus™(2lasers)
Fluorescein	488	YES	YES	YES	YES
Phycoerythrin(PE)	488	YES	YES	YES	YES
PE-TexasRed	488	YES	YES	YES	YES
BDCy-Chrome(PE-Cy5)	488	YES	YES	YES	NO^$
PropidiumIodide	488&595	YES	YES	YES	YES
PeridininChlorophylProtein(PerCP)	488	YES	YES	YES^*	YES^*
TexasRed	595	NO	NO	NO	YES⁺⁺
Allophycocyanin(APC)	595&633	NO	YES	NO	YES^$
APC-Cy7	595&633	NO	YES^#	NO	YES

^*PerCPishighlysensitivetophotobleachingandmustbeusedwithlaserpower<150mW

⁺⁺Canonlybeusedwithadyelaser

^#Notrecommended(dull)

^$BDCy-ChromeandAPCcannotbesimultaneouslyusedoninstrumentslackingcross-beamcompensation.

References:

Loken,M.R.,1990.ImmunofluorescenceTechniquesinFlowCytometryandSorting,2ndEd.,Wiley.pp341-353.

Parks,D.,L.Herzenberg,andL.Herzenberg.1989.Flowcytometryandfluorescence-activatedcellsorting.FundamentalImmunology,SecondEdition.WilliamPaul,Ed.RavenPress,Ltd,NewYork.

Zola,H.1995.Detectionofcytokinereceptorsbyflowcytometry.InCurrentProtocolsinImmunology(J.Coligan,A.Kruisbeek,D.Margulies,E.Shevach,W.Strober,eds.)JohnWileyandSons,NewYork.Unit6.21.

Immunofluorescenceandcellsorting.InCurrentProtocolsinImmunology.(J.Coligan,A.Kruisbeek,D.Margulies,E.Shevach,W.Strober,eds)JohnWileyandSons,NewYork.Unit5.1-5.6.5.Shapiro,H.M.1988.PracticalFlowCytometry,2ndEd.Wiley-Liss,NewYork.

Avaluableforumfordiscussionofmanyaspectsofflowcytometryis"TheCytometryElectronicMailingList."Tosubscribe,contact:cyto-request@flowcyt.cyto.purdue.edu.

================ 蚂蚁淘在线 ================

免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容