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AAT Bioquest/Cell Meter™ Cell Viability Assay Kit *Blue Fluorescence with 405 nm Excitation*/22784/500 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 405/450 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellAnalysis CellCytotoxicity |
| Related | ViABIlityandProliferation CellViability BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparecells:
Plate100to10,000cells/wellinatissueculturemicroplatewithblackwallandclearbottom,andaddtestcompoundsintothecells.Incubateforadesiredperiodoftime(suchas24,48or96hours)ina37°C,5%CO2incubator.Forblankwells(mediumwithoutthecells),addthesameamountofcompoundbuffer.Thesuggestedtotalvolumeis100µLfora96-wellplate,and25µLfora384-wellplate.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforproliferationorcytotoxicityinduction.Forproliferationassays,usefewercells;forcytotoxicityassays,usemorecellstostartwith.
2.Preparedye-loADIngsolution:
2.1 Thawoneofeachkitcomponentatroomtemperaturebeforeuse.
2.2 MakeCytoCalcein™Violet450,AMstocksolution:Add20µLofDMSO(ComponentB)intothevialofCytoCalcein™Violet450,AM(ComponentA),andmixwell.
Note:20µLofCytoCalcein™Violet450,AMstocksolutionisenoughforoneplate.UnusedCytoCalcein™Violet450stocksolutioncouldbealiquotedandstoredat<-20oCforonemonthifthetubesaresealedtightly.Protectfromlightandavoidrepeatedfreeze-thawcycles.
2.3 MakeCytoCalceinViolet450,AMdye-loadingsolutionforonecellplate:Addthewholecontent(20µL)ofCytoCalceinViolet450,AMstocksolution(fromStep2.2)into10mLofAssayBuffer(ComponentC),andmixthemwell.Thisworkingsolutionisstableforatleast2hoursatroomtemperature.
Note:IfthecellssuchasCHOcellscontainorganic-aniontransporterswhichpromotetheleakageofthefluorescentdyeovertime,aprobenecidstocksolutionshouldbepreparedandaddedtotheloadingbufferatafinalin-wellworkingconcentrationrangingfrom1to2.5mM.Aliquotandstoretheunusedprobenecidstocksolutionat<-20oC.
3.Runthecellviabilityassay:
3.1 Treatcellswithtestcompoundsasdesired(fromStep1).
Note:Itisnotnecessarytowashcellsbeforeaddingcompounds.However,iftestedcompoundsareserumsensitive,growthmediumandserumfactorscanbeaspiratedawaybeforeaddingcompounds.Add100µL/well(96-wellplate)and25µL/well(384-wellplate)of1XHank’ssaltsolutionand20mMHepesbuffer(HHBS)orthebufferofyourchoiceafteraspiration.Alternatively,cellscanbegrowninaserum-freemedia.
3.2 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofdye-loadingsolution(fromStep2.3).
3.3 Incubatethedye-loadingplateatroomtemperatureor37oCfor1hour,protectedfromlight.(Theincubationtimecouldbefrom15minutestoovernight.Wegottheoptimalresultswiththeincubationtimelessthan4hours.)
Note1:Theappropriateincubationtimedependsontheindividualcelltypeandcellconcentrationused.Optimizetheincubationtimeforeachexperiment.
Note2:DONOTwashthecellsafterloading.
Note3:Fornon-adherentcells,itisrecommendedtocentrifugecellplatesat800rpmfor2minuteswithbrakeoffafterincubation.
3.4 MonitorthefluorescenceintensityatEx/Em=405/460nm.
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