| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 646/659 |
| MW | ~500 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | CellBIOLOGy LabelingCells |
| Related | ViABIlityandProliferation CellViability MDRResearch BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparecells:
Plate100to10,000cellsperwellina96-wellblackwall/clearbottommicroplate.Addtestcompoundsintothecellsforadesiredperiodoftime(suchas24,48or96hours)ina37°C,5%CO2incubator.Forblankwells(mediumwithoutthecells),addthecorrespondingamountofcompoundbuffer.Thetotalsuggestedvolumeis100µLfora96-wellplate,and25µLfora384-wellplate.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforproliferationorcytotoxicityinduction.Forproliferation,usefewercells,andfortoxicityassays,usemorecellstostartwith.
2.PrepareCalceinDeepRed™workingsolution(for1plate):
2.1 Preparea2to5mMstocksolutionofCalceinDeepRed™inhigh-quality,anhydrousDMSO.Thestocksolutionshouldbeusedpromptly;anyremainingsolutionshouldbealiquotedandrefrozenat<-20oC.
Note:Avoidrepeatedfreeze-thawcycles,andprotectfromlight.
2.2 PrepareCalceinDeepRed™workingsolution:Onthedayoftheexperiment,eitherdissolveCalceinDeepRed™solidinDMSOorthawanaliquotoftheCalceinDeepRed™stocksolutiontoroomtemperature.Preparea5to10µMCalceinDeepRed™workingsolutioninHanksand20mMHepesbuffer(HHBS)orbufferofyourchoicepH7,with2.5mMprobenicid.
Note1:10mLofCalceinDeepRed™workingsolutionisnotstableatroomtemperature;preparefreshbeforeuse.
Note2:Manycellscontainorganic-aniontransporters.Itishighlyrecommendedtoaddadditional2.5mMprobenecid,aninhibitoroforganic-aniontransporters,intheCalceinDeepRed™workingsolution.
3.Runthecellviabilityassay:
3.1 Treatcellswithtestcompoundsasdesired(fromStep1).
Note:Itisnotnecessarytowashcellsbeforecompoundaddition.However,iftestedcompoundsareserumsensitive,growthmediumandserumfactorscanbeaspiratedawaybeforeaddingcompounds,providedresidualvolumesaftertheaspiratestep.Alternatively,cellscanbegrowninserum-freemedia.
3.2 Removethemediumfromthecells.
Note:MediummustberemovedbeforedyeloADIng.
3.3 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofCalceinDeepRed™workingsolution(fromStep2.2).
3.4 IncubatetheCalceinDeepRed™dye-loadingplateatroomtemperatureor37oCfor1hr,protectedfromlight.
Note1:Theappropriateincubationtimedependsontheindividualcelltypeandcellconcentrationused.Optimizetheincubationtimeforeachexperiment.
Note2:Fornon-adherentcells,itisrecommendedtocentrifugecellplatesat800rpmfor2minuteswithbrakeoffaftertheincubationofthedye.
3.5 MonitorthefluorescencechangeatEx/Em=635/670nm.
Note:Ifthebackgroundishigh,replacetheCalceinDeepRed™workingsolution(fromStep3.4)withHHBScontaining2.5mMprobenicidbeforemonitoringthefluorescencesignal.
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