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AAT Bioquest/Cell Explorer™ Fixable Live Cell Tracking Kit *Green Fluorescence*/22621/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 495/515 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy LabelingCells |
| Related | FluorescenceImaging BiochemicalAssays |
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.Centrifugetheplatesat800rpmfor2minuteswithbrakeoffpriortotheexperiments.
Note1:Forflowcytometryexperiment,preparecellsin0.5mLwarmmediumorbufferofyourchoiceatadensityof5×105to1×106cells/mL.
Note2:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.PrepareTrackIt™Greenstainsolution:
2.1 Prepare1000XTrackIt™Greenstocksolution:Add100µLofDMSO(ComponentC)intothevialofTrackIt™Green(ComponentA)andmixwell.
Note:Theunusedportionof1000XTrackIt™stocksolutionshouldbestoredat-20oC.Avoidrepeatedfreeze/thawcycles.
2.2 PrepareTrackIt™Greenworkingsolution: Dilute1000XTrackIt™Greenstocksolution(fromStep2.1)intoAssayBuffer(ComponentB)at1:1000ratiotomakeTrackIt™Greenworkingsolution.
Note:ThefinalconcentrationoftheTrackIt™Greenshouldbeempiricallydeterminedfordifferentcelltypesand/orexperimentalconditions.Ingeneral,long-termstaining(morethanabout3days)ortheuseofrapidlydividingcellswillrequire1:500dilutiontodoublethedyeconcentration.Dyeatalowerconcentrationupto1:2000dilutionmaybeneededforshorterexperiments,suchasviabilityassays.Tomaintainnormalcellularphysiologyandreducepotentialartifacts,theconcentrationofthedyeshouldbekeptaslowaspossIBLe.
3.Stainthecells:
3.1 AddequalvolumeofTrackIt™Greenworkingsolution(fromStep2.2)intothecellwells.Forexample,for96-wellplate,add100µL/wellofTrackIt™Greenworkingsolutionintothecells.
3.2 Incubatethecellsina37oC,5%CO2incubatorfor15to30minutes.
3.3 WashcellswithHHBSoranappropriatebufferfor3times.
Note:Alternatively,fixthecellsatthispoint.Storethefixedcellsat4°C,andimagethecellslater.
3.4 ImagethecellsusingafluorescencemicroscopewithFITCfilters(Ex/Em=490/520nm).OrmonitorthefluorescenceintensitywithaflowcytometerusingtheFL1channel(Ex/Em=490/525nm),gateonthecellsofinterest,excludingdebris.
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