流式细胞技术荧光抗体

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1、Fluorochromes23-13536-00Rev.01FluorochromePropertiesDesirablepropertiesforfluorochromes:HighrelativebrightnessNarrowemissionspectrum(lowspectraloverlapincombination)Easilyconjugated(forimmunophenotyping)Fluorochromescanbecharacterizedby:TypeofmoleculeExcitati

2、onandemissionwavelengthsRelativebrightness223-13536-00Rev.01FluorochromeMoleculeTypesSmallorganicmoleculesexamples:FITC,BDHorizonTMV450,Cy7Fluorescentproteinsexamples:PE,APC,PerCPTandemdyes—typically,thecouplingofafluorescentproteindonorwithasmallorganicmolecu

3、leacceptorexamples:PE-Cy7,PerCP-CyTM5.5Nanocrystals(Qdots)—inorganicsemiconductorsexamples:Qdot565,Qdot605323-13536-00Rev.01SomeCommonFluorochromesFluorochromeTypeofmoleculeFluoresceinisothyocyanate(FITC)SmallorganicAlexaFluor®488SmallorganicPhycoerythrin(PE)P

4、roteinPE-CyTM5ProteintandemPeridininchlorophyllprotein(PerCP)ProteinPerCP-CyTM5.5ProteintandemPE-CyTM7ProteintandemAllophycocyanin(APC)ProteinAlexaFluor®647SmallorganicAPC-CyTM7ProteintandemBDTMAPC-H7ProteintandemBDHorizonTMV450SmallorganicPacificBlueTMSmallor

5、ganicBDHorizonTMV500SmallorganicAmCyanProtein423-13536-00Rev.01SmallOrganicFluorochromesAdvantagesLowmolecularweightEasytoconjugate—directattachmenttofreeaminogroupsonmAbExcellentstabilityExtremelyconsistentemissionspectraDisadvantagesSmallStokesShift(50−100nm

6、)Tendtobelessbright523-13536-00Rev.01ProteinFluorochromesAdvantagesGoodstabilityConsistentemissionspectraMediumStokesShift(75−200nm)TendtobemorebrightDisadvantagesHighmolecularweightMoredifficulttoconjugate—intermediariesneededtoattachtomAb623-13536-00Rev.01Ta

7、ndemDyeFluorochromesAdvantagesVerylargeStokesShift(150−300nm)Tendtobeverybright—oftenbrighterthanthefluorescentproteindonorDisadvantagesHighmolecularweight(similartofluorescentprotein)Difficulttomakeconsistently(lot-to-lotvariationinemissionproperties)Harderto

8、conjugate(sameasfluorescentprotein)Sometandemshavepoorstability723-13536-00Rev.01NanocrystalFluorochromesAdvantagesLargeStokesShift(100−500nm)TendtobeverybrightEmissionpeaksarecons