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AAT Bioquest/Cell Meter™ Cell Viability Assay Kit *Blue Fluorescence*/22785/500 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 360/445 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellAnalysis CellCytotoxicity |
| Related | ViABIlityandProliferation CellViability BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
Plate100to10,000cells/wellinatissueculturemicroplatewithblackwallandclearbottom,andaddtestcompoundsintothecells.Incubateforadesiredperiodoftime(suchas24,48or96hours)ina37°C,5%CO2incubator.Forblankwells(mediumwithoutthecells),addthesameamountofcompoundbuffer.Thesuggestedtotalvolumeis100µLfora96-wellplate,and25µLfora384-wellplate.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforproliferationorcytotoxicityinduction.Forproliferationassays,usefewercells;forcytotoxicityassays,usemorecellstostartwith.
2.1Thawoneofeachkitcomponentatroomtemperaturebeforeuse.
2.2MakeCytoCalcein™Blue,AMstocksolution:Add20µLofDMSO(ComponentB)intothevialofCytoCalcein™Blue,AM(ComponentA),andmixwell.
Note:20µLofCytoCalcein™Blue,AMstocksolutionisenoughforoneplate.UnusedCytoCalcein™Blue,AMstocksolutioncouldbealiquotedandstoredat<-20=""°c=""for=""one=""month=""if=""the=""tubes=""are=""sealed=""tightly.=""protect=""from=""light=""and=""avoid=""repeated=""freeze-thaw="">
2.3MakeCytoCalceinBlue,AMdye-loadingsolutionforonecellplate:Addthewholecontent(20µL)ofCytoCalceinBlue,AMstocksolution(fromStep2.2)into10mLofAssayBuffer(ComponentC),andmixthemwell.Thisworkingsolutionisstableforatleast2hoursatroomtemperature.
Note:IfthecellssuchasCHOcellscontainorganic-aniontransporterswhichpromotetheleakageofthefluorescentdyeovertime,aprobenecidstocksolutionshouldbepreparedandaddedtotheloadingbufferatafinalin-wellworkingconcentrationrangingfrom1to2.5mM.Aliquotandstoretheunusedprobenecidstocksolutionat<-20="">
3.1Treatcellswithtestcompoundsasdesired(fromStep1).
Note:Itisnotnecessarytowashcellsbeforeaddingcompounds.However,iftestedcompoundsareserumsensitive,growthmediumandserumfactorscanbeaspiratedawaybeforeaddingcompounds.Add100µL/well(96-wellplate)and25µL/well(384-wellplate)of1XHank"ssaltsolutionand20mMHepesbuffer(HHBS)orthebufferofyourchoiceafteraspiration.Alternatively,cellscanbegrowninaserum-freemedia.
3.2Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofdye-loadingsolution(fromStep2.3).
3.3Incubatethedye-loadingplateatroomtemperatureor37°Cfor1hour,protectedfromlight.(Theincubationtimecouldbefrom15minutestoovernight.Wegottheoptimalresultswiththeincubationtimelessthan4hours.)
Note1:Theappropriateincubationtimedependsontheindividualcelltypeandcellconcentrationused.Optimizetheincubationtimeforeachexperiment.
Note2:DONOTwashthecellsafterloading.
Note3:Fornon-adherentcells,itisrecommendedtocentrifugecellplatesat800rpmfor2minuteswithbrakeoffafterincubation.
3.4MonitorthefluorescenceintensityatEx/Em=360/450nm.
| References&Citations |  CitationExplorer |
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ToxicokineticsandtoxicodynamicsofchlorpyrifosisalteredinembryosofJapanesemedakaexposedtooilsandsprocess-affectedwater:evidenceforinhibitionofP-glycoprotein
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Journal:JournalofAppliedToxicology(2016)
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