>
产品中心 >
Fluorescent_dyes >
AAT Bioquest/Cell Meter™ Live Cell Caspase 3/7 and Phosphatidylserine Detection Kit *Triple Fluorescence Colors*/22850
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | None/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellAnalysis CellCytotoxicity |
| Related | ApoptosisandCytotoxicity CellApoptosis BiochemicalAssays |
1. Culturecellstoadensityoptimalforapoptosisinductionaccordingtoyourspecificinductionprotocol,butnottoexceed2x106cells/mL(ornottoexceed3x105cells/100μL/wellina96-wellblackclear-bottomplate).Atthesametime,cultureanon-inducednegativecontrolcellpopulationatthesamedensityastheinducedpopulationforeverylabelingcondition. Hereareafewexamplesfor inducingapoptosisinsUSPensionculture:
1)TreatingJurkatcellswith2μg/mlcamptothecinfor3hours.
2)TreatingJurkatcellswith1μMstaurosporinefor3hours.
3)TreatingHL-60cellswith4μg/mlcamptothecinfor4hours.
4)TreatingHL-60cellswith1μMstaurosporinefor4hours.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforapoptosisinduction.Foradherentcells,use2-3x104cells/welltostart.
2. Make150XTF3-DEVD-FMKDMSOstocksolutionbyadding200μLofDMSOtothevialofTF3-DEVD-FMK(ComponentA).
3. AddTF3-DEVD-FMKata1:150ratioand/orAnnexinV-iFluor488™(ComponentB)at1:100ratiointoeachwell,incubatethecellsina37°C,5%CO2incubatorfor1hour.
Note1:Thecellscanbeconcentratedupto~5X106cells/mLforTF3-DEVD-FMKlabeling.Theunused150XTF3-DEVD-FMKDMSOstocksolutionshouldbedividedassingleusealiquotandstoredat-20°C.
Note2:Foradherentcells,gentlyliftthecellswith0.5mMEDTAtokeepthecellsintact,andwashthecellsoncewithserum-containingmediapriortoincubationwithTF3-DEVD-FMK.
Note3:AnnexinVflowcytometricanalysisonadherentcellsisnotroutinelytestedsincespecificmembranedamagemayoccurduringcelldetachmentorharvesting.However,methodsforutilizingAnnexinVforflowcytometryonadherentcelltypeshavebeenpreviouslyreportedbyCasiola-Rosenetal.andvanEngelendetal(seeRefs1and2).
Note4:Theappropriateincubationtimedependsontheindividualcelltypeandcellconcentrationused.Optimizetheincubationtimeforeachexperiment.
4. Ifdesired,labelthecellswithaDNAstain(suchasHoechstforwholepopulationofthecellnucleusstain,orpropidiumiodidefordeadcellsifthecellslabelwithAnnexinV-iFluor488™only).
5. Spindownthecellsat~200gfor2minutes,andwashcellswithandwashcellswith1mL(or200μL/wellifusing96-wellplate)washbuffer(ComponentE)twice.Resuspendthecellsindesiredamountofwashingbuffer.
Note1:TF3-DEVD-FMKand AnnexinV-iFluor488™ arefluorescent,thusitisimportanttowashoutanyunboundreagenttoeliminatethebackground.
Note2:Fordetachedcells,theconcentrationofcellsshouldbeadjustedto2-5X105cells/100μLaliquotpermicrotiterplatewellforuseinstep6.
6. Monitorthefluorescenceintensitybyfluorescencemicroscopy,flowcytometer,orfluorescentmicroplatereaderatEx/Em=550/595nmforTF3-DEVD-FMK,490/525forAnnexinV-iFluor488™,350/461nmforHoechststain,and535/635forpropidiumiodide.
6.1 Forflowcytometry,monitorthefluorescenceintensityusingtheFL1channelforAnnexinV-iFluor488™,FL2channelforTF3-DEVD-FMK.Gateonthecellsofinterest,excludingdebris.
6.2 Forfluorescencemicroscopyandfluorescentmicroplatereader.Place100μLofthecellsuspensionsintoeachofwellsofa96-wellblackmicrotiterplate.
Note: Ifitisnecessarytoequilibratethecellconcentrations,adjustthesuspensionvolumefortheinducedcellstoapproximatethecelldensityofthenon-inducedpopulation. Thisadjustmentstepisoptionalifyourcelltreatmentdoesnotresultinadramaticlossinstimulatedcellpopulationnumbers.
6.3 ObservecellsunderafluorescencemicroscopeusingTRITCchannelforTF3-DEVD-FMK,and/orFITCchannelforAnnexinV-iFluor488™(TRITCchannelforpropidiumiodidestaining,DAPIchannelforHoechststaining).
6.4 MonitorthefluorescenceintensityusingEx/Em=490/525nm(cutoffat515nm)forTF3-DEVD-FMK,and/or550/595nm(cutoff575)forAnnexinV-iFluor488™ bottomreadmodeforafluorescentmicroplatereader.
| References&Citations |  CitationExplorer |
Anthocyanin-richblackcurrantextractinhibitsproliferationoftheMCF10AhealthyhumanbreastepithelialcelllinethroughinductionofG0/G1arrestandapoptosis
Authors:NaokiNanashima,KayoHorie,MitsuruChiba,ManabuNakano,HayatoMaeda,ToshiyaNakamura
Journal:MolecularMedicineReports(2017):6134--6141
ClusterinsignalsviaApoER2/VLDLRandinducesmeiosisofmalegermcells
Authors:MuhammadAssadRiaz,AngelikaStammler,MareikeBorgers,LutzKonrad
Journal:AmericanJournalofTranslationalResearch(2017):1266
DetectingApoptosis,Autophagy,andNecrosis
Authors:JackColeman,RuiLiu,KathyWang,ArunKumar
Journal:ApoptosisMethodsinToxicology(2016):77--92
