MP06999DiIDerivativesforLong-TermCellularLabelingProductInformationRevised:09–August–2005DiIDerivativesforLong-TermCellularLabelingIntroductionThestrong,photostablefluorescence,excellentcellularretention,andminimalcytotoxicityofDiIandotherlong-chaincarbocyaninemembraneprobesmakethemparticularlysuitableforlong-termlabelingandtrackingofcells,1-4intracellularmem-branes,5,6liposomes,7viruses,8andlipoproteins.9,10Unfortunately,currentlyavailablelipophiliccarbocyanines,includingDiIandDiOandthePKH2andPKH26dyes,11,12sometimesrequirethepresenceofosmolarityregulatingagentsortheabsenceofsaltstoavoiddyeprecipitationfromthestainingmediumduringlabelingofcellsuspensions.Furthermore,DiIlabelingisnotcompatiblewithlong-termpreservationusingstandardtissuefixationpro-cedures.Tocircumventtheselimitations,MolecularProbeshasdevelopedseverallipophiliccarbocyaninederivativeswithim-provedwatersolubilityandstainingpersistenceafterfixation:•Chloromethylbenzamido(CellTrackerTMCM-DiI;C7000,C7001,N22883,V22888)andmethylbenzamido(FM®-DiI,F6999)derivativesofDiI•SulfonatedDiIderivativesSP-DiIC18(3)(D7777)andDiIC18(3)-DS(D7776)•SulfonatedderivativesofDiO(SP-DiOC18(3);D7778)andthered-fluorescentcarbocyaninetracerDiD(DiIC18(5)-DS;D12730)CM-DiIincorporatesamildlythiol-reactivechloromethylsubstituentthatconfersaldehydefixabilityviaconjugationtothiol-containingpeptidesandproteins.UnlikeDiIandPKH26,CM-DiIisretainedincellsthroughoutfixation,permeabilization,andparaffinembeddingprocedures.13,14Labelingwithsulfonatedcarbocyaninetracersalsoappearstobecompatiblewithaldehydefixationandacetonepermeabiliza-tiontreatments,althoughthemechanismresponsibleforthepersistenceoflabelingisnotcurrentlyknown.WehaveobservedexcellentretentionofSP-DiIC18(3)andSP-DiOC18(3)staininginhumanBcellsunderconditionsinwhichvirtuallyallofthePKH26dyeislostduringthelipidextractionstep(Figure1).Furthermore,wehaveobservedthatinsomecasesacetonetreatmentactuallyenhancesthefluorescenceofcellsstainedwithSP-DiIC18(3)andSP-DiOC18(3)(partic...
