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AAT Bioquest/Live or Dead™ Cell Viability Assay Kit *Green/Red Dual Fluorescence*/22789/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 492/515 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellAnalysis CellCytotoxicity |
| Related | ViABIlityandProliferation CellViability BiochemicalAssays |
1.Preparecells:
Plate100to100,000cells/wellinatissueculturemicroplatewithblackwallandclearbottom.Addtestcompoundsintothecellsandincubateforadesiredperiodoftime(suchas24,48or96hours)ina37°C,5%CO2incubator.Forblankwells(mediumwithoutthecells),addthesameamountofcompoundbuffer.Thesuggestedtotalvolumeis100µL/well/96-wellplateand25µL/well/384-wellplate.
Note:Eachcelllineshouldbeevaluatedontheindividualbasistodeterminetheoptimalcelldensityforproliferationorcytotoxicityinduction.Forproliferationassays,usefewercells,andforcytotoxicityassays,usemorecellstostartwith.
2.Preparedye-loadingsolution:
2.1 Thawoneofeachkitcomponentatroomtemperaturebeforeuse.
2.2 MakeCytoCalcein™Greenstocksolution:Add20µLofDMSO(ComponentC)intothevialofCytoCalcein™Green(ComponentA),andmixthemwell.
Note:20µLofCytoCalcein™Greenstocksolutionisenoughforoneplate.UnusedCytoCalcein™Greenstocksolutioncanbealiquotedandstoredat<-20oCforonemonthifthetubesaresealedtightly.Protectfromlightandavoidrepeatedfreeze-thawcycles.
2.3 MakeCytoCalcein™Green/PropidiumIodidedye-loadingsolutionforonecellplate:Addthewholecontent(20µL)ofCytoCalcein™Greenstocksolution(fromStep2.2)and20µLPropidiumIodide(20ComponentB)into10mLofAssayBuffer(ComponentC),andmixwell.Theworkingsolutionisstableforatleast2hoursatroomtemperature.
Note1:IfthecellssuchasCHOcellscontainorganic-aniontransporterswhichcausetheleakageofthefluorescent
dyeovertime,aprobenecidstocksolutionshouldbepreparedandaddedtotheloadingbufferatafinalin-well
workingconcentrationrangingfrom1to2.5mM.Aliquotandstoretheunusedprobenecidstocksolutionat<-20oC.
Note2:Astheoptimalstainingconditionsmayvarydependingondifferentcelltypes,it’srecommendedtodeterminetheappropriateconcentrationofComponentAandBindividually.
3.Runthecellviabilityassaywithplatereaderorfluorescencemicroscope:
3.1 Treatcellswithtestcompoundsasdesired(fromStep1).
Note:Itisnotnecessarytowashcellsbeforeaddingcompound.However,iftestedcompoundsareserumsensitive,growthmediumandserumfactorscanbeaspiratedawaybeforeaddingcompounds.Add100µL/well/96-wellplateand25µL/well/384-wellplateof1XHank’ssaltsolutionand20mMHepesbuffer(HHBS)orthebufferofyourchoiceafteraspiration.Alternatively,cellscanbegrowninserum-freemedia.
3.2 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofdye-loadingsolution(fromStep2.3).
3.3 Incubatethedye-loadingplateatroomtemperatureor37oCfor30minutesto1hour,protectedfromlight.(Theincubationtimecouldbefrom15minutestoovernight.Wegottheoptimalresultswiththeincubationtimelessthan4hours.)
Note1:Theappropriateincubationtimedependsontheindividualcelltypeandcellconcentrationused.Optimizetheincubationtimeforeachexperiment.
Note2:DONOTwashthecellsafterloading.
Note3:Fornon-adherentcells,itisrecommendedtocentrifugecellplatesat800rpmfor2minuteswithbrakeoffafterincubation.
3.4 MonitorthefluorescenceintensityatEx/Em=490/525nm(FITCfilter)forlivecells,and540/620nm(TRITCfilter)fordeadcellswithabottomreadmode.
4.Runthecellviabilityassaywithaflowcytometer:
4.1 Treatcellswithtestcompoundsforadesiredperiodoftime.
4.2 Centrifugethecellstoget1-5×105cells/tube.
4.3 Resuspendcellsin500μLofCytoCalcein™Green/PropidiumIodidedye-loadingsolution(fromStep2.3).
4.4 Incubateatroomtemperatureor37°Cfor10to30minutes,protectedfromlight.
Optional:WashthecellswithHHBSorbufferofyourchoice.Resuspendcellsin500μLofHHBStoget1-5×105cellspertube.
4.5 MonitorthefluorescenceintensityatEx/Em=490/525and620nmwithaflowcytometer(usingFL1andFL2channels).
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