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Fluorescent_dyes >
AAT Bioquest/Cell Navigator™ Live Cell Endoplasmic Reticulum (ER) Staining Kit *Red Fluorescence*/22636/100 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 589/620 |
| 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/90μLfora96-wellplateor2,500to10,000cells/well/20μLfora384-wellplate.
1.2. Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandsUSPendthecellpelletsinculturemediumat50,000-100,000cells/well/90µLfora96-wellpoly-Dlysineplateor10,000-25,000cells/well/20µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortoyourexperiment.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.Prepareworkingsolution:
2.1 Thawkitcomponentsatroomtemperaturebeforeuse.
2.2 MakeERRed™500Xstocksolution:Add20µLofDMSO(ComponentC)intoERRed™(ComponentA)tomake500Xstocksolution.
2.3 MakeERRed™workingsolution:Add20µLof500Xstocksolution(ComponentA)into10mLofLiveCellStainingBuffer(ComponentB),andmixwell.Theworkingsolutionisstableforatleast2hoursatroomtemperature.
Note:20µLof500XERRed™stocksolution(ComponentA)isenoughforone96-wellplate.UnusedERRed™500Xstocksolutioncanbealiquotedandstoredat≤-20ºCfortwoweeksifthetubesaresealedtightly.Protectfromlightandavoidrepeatedfreeze-thawcycles.
3.Staincells:
3.1 Add100µL/well(96-wellplate)or50µL/well(384-wellplate)ofERRed™workingsolution(fromStep2.3)inthecellplate.Incubatecellswithworkingsolutionat37ºCfor15-30minutes,protectedfromlight.
Note:TheoptimalconcentrationoftheERprobevariesdependingonthespecificapplication.Concentrationhigherthantheworkingsolutioncanbetoxictocells.ThestainingconditionsmaybemodifiedaccordingtotheparticularcelltypeandthepermeABIlityofthecellsortissuestotheprobe.
3.2 Removeworkingsolutionineachwell.Washcellswithphysicallyrelevantbufferthreetimes.
3.3 Fixcellsafterstaining(Optional).Fixthecellswith4%formaldehydefor5-10minutes.Washcellswithphysicallyrelevantbufferthreetimes.
3.4 ObservethefluorescencesignalincellsusingfluorescencemicroscopewithaTRITCorCy3filterset.
