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AAT Bioquest/Cell Explorer™ Live Cell Labeling Kit *Red Fluorescence*/22609/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 646/659 |
| 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/100µLfor96-wellplatesor2,500to10,000cells/well/25µLfor384-wellplates.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletsinculturemediumat50,000-100,000cells/well/100µLfor96-wellpoly-Dlysineplatesor10,000-25,000cells/well/25µLfor384-wellpoly-Dlysineplates.Centrifugetheplatesat800rpmfor2minuteswithbrakeoffpriortotheexperiment.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.PrepareCalceinRed™stainsolution:
2.1 PrepareCalceinRed™stocksolution:Add20µLofDMSOintoCalceinRed™vial(ComponentA)andmixthemwell.
Note:20µLofCalceinRed™stocksolutionisenoughfor1plate.UnusedCalceinRed™stocksolutioncanbealiquotedandstoredat<-20oCfor2weeksifthetubesaresealedtightly.Avoidrepeatedfreeze-thawcyclesandprotectfromlight.
2.2 PrepareCalceinRed™workingsolutionforonecellplate:Add20µLofDMSOreconstitutedCalceinRed™stocksolution(fromStep2.1)into10mLofHHBS(ComponentB),andmixthemwell.
3.Stainthecells:
3.1 Removethegrowthmediumfromthecellplates.
Note:Itisimportanttoremovethegrowthmediuminordertominimizethebackgroundfluorescenceandincreasethesignaltobackgroundratio.
3.2 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)CalceinRed™workingsolution(fromStep2.2)intothecellplate.
3.3 Incubatethecellsina37oC,5%CO2incubatorfor30minutesto2hours.
3.4 RemovetheCalceinRed™workingsolutionfromthecells,andwashthecellswithHHBS(ComponentB)for2to3times,andreplacewithHHBS.
3.5 ImagethecellsusingafluorescencemicroscopewithCy5filters(Ex/Em=646/660nm).
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