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AAT Bioquest/Calcein, AM *UltraPure grade* *CAS 148504-34-1*/22003/1 mg
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 495/515 |
| MW | 994.86 |
| CAS# | 148504-34-1 |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | CellBIOLOGy LabelingCells |
| Related | ViABIlityandProliferation CellViability MDRResearch |
| Spectrum | AdvancedSpectrumViewer |
AMestersarethenon-polarestersthatreadilycrosslivecellmembranes,andrapidlyhydrolyzedbycellularesterasesinsidelivecells.AMestersarewidelyusedforloadingavarietyofpolarfluorescentprobesintolivecellsnon-invasively.However,cautionsmustbeexcisedwhenAMestersareusedsincetheyaresusceptibletohydrolysis,particularlyinsolution.Theyshouldbereconstitutedjustbeforeuseinhigh-quality,anhydrousdimethylsulfoxide(DMSO).DMSOstocksolutionsmaybestoreddesiccatedat‑20°Candprotectedfromlight.Undertheseconditions,AMestersshouldbestableforseveralmonths.
FollowingisourrecommendedprotocolforloadingAMestersintolivecells.Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.
a) Preparea2to5mMstocksolutionofAMestersinhigh-quality,anhydrousDMSO.ThenonionicdetergentPluronic®F-127issometimesusedtoincreasetheaqueoussolubilityofAMesters.
Note:Anequalvolumeof20%Pluronic®F-127solutioncanbeaddedtoDMSOstocksolutionsofcellviabilityindicatorbeforedilutingintotheloadingbuffer.ThefinalPluronic®F-127concentrationisabout0.02%.AvarietyofPluronic®F-127solutionscanbepurchasedfromAATBioquest.
Caution:Thelong-termstorageofAMestersinthepresenceofPluronic®F-127isnotrecommended.
b) Onthedayoftheexperiment,eitherdissolvethesolidindicatorsofcellviabilityinDMSOorthawanaliquotoftheindicatorstocksolutionstoroomtemperature.Prepareaworkingsolutionof1to10µMinthebufferofyourchoice(suchasHanksandHepesbuffer).Formostofthecelllines,cellviabilityindicatorsatthefinalconcentrationrangingfrom4to5µMarerecommended.Theexactconcentrationofindicatorsrequiredforcellloadingmustbedeterminedempirically.
c) Ifyourcellscontainingtheorganicanion-transports,probenecid(1–2.5mM)orsulfinpyrazone(0.1–0.25mM)maybeaddedtothecellmediumtoreduceleakageofthede-esterifiedindicators.IncubatecellswithAMestersatroomtemperatureor37°Cfor20minutestoonehour.
Note:Decreasingtheloadingtemperaturemightreducetheindicatorcompartmentalization.
d) Washcellsincellviabilityindicator-freebuffer(containingananiontransporterinhibitor,ifapplicable)toremoveexcessprobes.
Note:Cellsampleswereimmediatelyanalyzedbyflowcytometrytodeterminetheaveragefluorescencepercellattimezerowithoutwashing.
e) RuntheexperimentsatthedesiredEx/Emwavelengths(seeTable1).
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