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AAT Bioquest/Cell Explorer™ Live Cell Tracking Kit *Blue Fluorescence*/22620/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 403/445 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy LabelingCells |
| Related | FluorescenceImaging BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.PrepareCells:
1.1 Foradherentcells:Platecellsovernightingrowthmediumat10,000to40,000cells/well/90µLfor96-wellplatesor2,500to10,000cells/well/20µLfor384-wellplates.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletsinculturemediumat50,000-100,000cells/well/90µLfor96-wellpoly-Dlysineplatesor10,000-25,000cells/well/20µLfor384-wellpoly-Dlysineplates.Centrifugetheplatesat800rpmfor2minuteswithbrakeoffpriortotheexperiment.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.PrepareTrackIt™Bluestainsolution:
2.1 Prepare2mMTrackIt™Bluestocksolution:Add25µLofDMSO(ComponentB)intooneoftheTrackIt™Bluevials(ComponentA)tomake2mMstocksolution.
Note:TheunusedportionoftheTrackIt™stocksolutionshouldbestoredat-20oC.Avoidrepeatedfreeze/thawcycles.
2.2 Prepare10XTrackIt™Blueworkingsolution:Dilute2mMofTrackIt™Bluestocksolution(fromStep2.1)intoAssayBuffer(ComponentC)tomake5to50µMTrackIt™Blueworkingsolution.Theworkingsolutionshouldbepreparedenoughforallthewellsat10μL/wellwiththeappropriateconcentration.Forexample,togetTrackIt™Blueatthefinalconcentrationof20μMforone96-wellmicroplate,dilute10μLoftheTrackIt™Bluestocksolutioninto1mLofAssayBuffer(ComponentC)tomake1mLof20μM(10X)TrackIt™Blueworkingsolution.
Note1:ThefinalconcentrationoftheTrackIt™Blueshouldbeempiricallydeterminedfordifferentcelltypesand/orexperimentalconditions.Itisrecommendedtotestattheconcentrationsthatareatleastoveratenfoldrange.
Note2:Wefoundthat2uMfinalinwellconcentrationissufficientformostofcelllines.
3.Stainthecells:
3.1 Tothecellwellsadd10XTrackIt™Blueworkingsolution(fromStep2.2),whichshouldbeequalto1/10ofthevolumeofcellculturemedium.Forexample,fora96-wellplate,add10µL/wellof10XTrackIt™Blueworkingsolutionintothecells.
3.2 Incubatethecellsina37oC,5%CO2incubatorfor15minto1hour.
3.3 WashcellswithHanksand20mMHepesbuffer(HHBS)oranappropriatebuffer.
3.4 Fillthecellwellswithgrowthmedium.
3.5 AnalyzethecellsusingafluorescencemicroscopeorflowcytometerwithDAPIfiltersets(Ex/Em=360/445nm).
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