| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 560/574 |
| 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.Addtestcompoundsintothecellsandincubateforadesiredperiodoftime(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™Redstocksolution:Add20µLofDMSO(ComponentB)intoonevialofCytoCalcein™Red(ComponentA),andmixwell.
Note:20µLofCytoCalcein™Redstocksolutionisenoughforoneplate.UnusedCytoCalcein™Redstocksolutioncanbealiquotedandstoredat<-20oCforonemonthifthetubesaresealedtightly.Protectfromlightandavoidrepeatedfreeze-thawcycles.
2.3 MakeCytoCalcein™Reddye-loadingsolutionforonecellplate:Addthewholecontent(20µL)ofCytoCalcein™Redstocksolution(fromStep2.2)into10mLofAssayBuffer(ComponentC),andmixwell.Theworkingsolutionisnotstable,useitpromptly.
Note:Ifthecells,suchasCHOcells,containorganic-aniontransporterswhichcausetheleakageofthefluorescentdyeovertime,aprobenecidstocksolutionshouldbepreparedandaddedtotheloadingbufferatafinalin-wellworkingconcentrationof1-2.5mM.Aliquotandstoretheunusedprobenecidstocksolutionat<-20oC.
3.Runthecellviabilityassay:
3.1 Treatcellswithtestcompoundsasdesired(fromStep1).
Note:Itisnotnecessarytowashcellsbeforeaddingcompound.However,iftestedcompoundsareserumsensitive,growthmediumandserumfactorscanbeaspiratedawaybeforeaddingcompounds.Add100µL/well(96-wellplate)and25µL/well(384-wellplate)of1XHank’ssaltsolutionand20mMHepesbuffer(HHBS)orthebufferofyourchoiceafteraspiration.Alternatively,cellscanbegrowninserum-freemedia.
3.2 Removegrowthmedium.
3.3 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofdye-loadingsolution(fromStep2.3).
3.4 Incubatethedye-loadingplateatroomtemperatureor37oCfor30min-1hour,protectedfromlight.
Note1:Theappropriateincubationtimedependsontheindividualcelltypeandcellconcentrationused.Optimizetheincubationtimeforeachexperiment.
Note2:Fornon-adherentcells,itisrecommendedtocentrifugecellplatesat800rpmfor2minuteswithbrakeoffafterincubation.
3.5 MonitorthefluorescenceintensityatEx/Em=540/590nm(cutoffat570nm)withbottomreadmode.
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