23-13536-00Rev.01Fluorochromes1FluorochromePropertiesDesirablepropertiesforfluorochromes:•Highrelativebrightness•Narrowemissionspectrum(lowspectraloverlapincombination)•Easilyconjugated(forimmunophenotyping)Fluorochromescanbecharacterizedby:•Typeofmolecule•Excitationandemissionwavelengths•Relativebrightness23-13536-00Rev.01Fluorochromes2FluorochromeMoleculeTypes•Smallorganicmoleculesexamples:FITC,BDHorizonTMV450,Cy7•Fluorescentproteinsexamples:PE,APC,PerCP•Tandemdyes—typically,thecouplingofafluorescentproteindonorwithasmallorganicmoleculeacceptorexamples:PE-Cy7,PerCP-CyTM5.5•Nanocrystals(Qdots)—inorganicsemiconductorsexamples:Qdot565,Qdot60523-13536-00Rev.01Fluorochromes3SomeCommonFluorochromesFluorochromeTypeofmoleculeFluoresceinisothyocyanate(FITC)SmallorganicAlexaFluor®488SmallorganicPhycoerythrin(PE)ProteinPE-CyTM5ProteintandemPeridininchlorophyllprotein(PerCP)ProteinPerCP-CyTM5.5ProteintandemPE-CyTM7ProteintandemAllophycocyanin(APC)ProteinAlexaFluor®647SmallorganicAPC-CyTM7ProteintandemBDTMAPC-H7ProteintandemBDHorizonTMV450SmallorganicPacificBlueTMSmallorganicBDHorizonTMV500SmallorganicAmCyanProtein23-13536-00Rev.01Fluorochromes4SmallOrganicFluorochromesAdvantages•Lowmolecularweight•Easytoconjugate—directattachmenttofreeaminogroupsonmAb•Excellentstability•ExtremelyconsistentemissionspectraDisadvantages•SmallStokesShift(50−100nm)•Tendtobelessbright23-13536-00Rev.01Fluorochromes5ProteinFluorochromesAdvantages•Goodstability•Consistentemissionspectra•MediumStokesShift(75−200nm)•TendtobemorebrightDisadvantages•Highmolecularweight•Moredifficulttoconjugate—intermediariesneededtoattachtomAb23-13536-00Rev.01Fluorochromes6TandemDyeFluorochromesAdvantages•VerylargeStokesShift(150−300nm)•Tendtobeverybright—oftenbrighterthanthefluorescentproteindonorDisadvantages•Highmolecularweight(similartofluorescentprotein)•Difficulttomakeconsistently(lot-to-lotvariationinemissionproperties)•Hardertoconjugate(sameasfluorescentprotein)•Sometandemshavepoorstability23-13536-00Rev.01Fluorochromes7NanocrystalFluorochromesAdvantages•LargeStokesShift(100−500nm)•Tendtobeverybright•Emissionpeaksareconsistentandnarrow,anddonotchangewithvariationsintheexcitationsource•Highlyresistanttophotobleaching•NanocrystalssharebiophysicalandconjugationpropertiesDisadvantages•Difficulttoconjugate•Instabilityofbindings•Cytotoxicity•Wideexcitationrangeproducescross-laserspillover23-13536-00Rev.01Fluorochromes8ExcitationandEmission•Excitationwavelengthsdeterminelasersthatcanexcitethefluorochrome.•EmissionwavelengthsdeterminefiltersandPMTsthatcanmeasuretheemissionsignal.23-13536-00Rev.01Fluorochromes9KnowYourCytometerCytometerConfiguration•BDFACSCantoTMII4-2-2configurationisshownbelow•BDTMLSRII4-2-2configurationissimilar4detectorsforbluelaser2detectorsforredlaser2detectorsforvioletlaser23-13536-00Rev.01Fluorochromes10TypicalExcitationandEmissionBDFACSCantoII4-2-2(BDLSRII4-2-2issimilar)DetectorRangeVioletLaser405nmBlueLaser488nmRedLaser633nm410–490nmBDHorizonV450PacificBlueTM500–560nmBDHorizonV500AmCyan515–545nmFITCAlexaFluor®488564–606nmPE650–670nmAPCAlexaFluor®647670–735nmPerCP-Cy5.5PE-Cy5PerCP750–810nmPE-Cy7BDAPC-H7APC-Cy723-13536-00Rev.01Fluorochromes11FluorochromeUseDependsontheCytometerConfiguration6-color8-colorMorethan8colorsFITC,AlexaFluor®488FITC,...
