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Fluorochrome Absorption and Emission Spectra
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. 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 *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.
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