| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 492/514 |
| MW | N/A |
| CAS# | N/A |
| Solvent | Water |
| Storage | F/D/L |
| Category | CellAnalysis CellApoptosis |
| Related | ApoptosisandCytotoxicity BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1. Culturecellstoadensityoptimalforapoptosisinductionaccordingtoyourspecificinductionprotocol,butnottoexceed2x106cells/mL.Atthesametime,cultureanon-inducednegativecontrolcellpopulationatthesamedensityastheinducedpopulationforeverylabelingcondition. Hereareafewexamplesfor inducingapoptosisinsUSPensionculture:
1)TreatingJurkatcellswith2μg/mlcamptothecinfor3hours.
2)TreatingJurkatcellswith1μMstaurosporinefor3hours.
3)TreatingHL-60cellswith4μg/mlcamptothecinfor4hours.
4)TreatingHL-60cellswith1μMstaurosporinefor4hours.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforapoptosisinduction.
2. Make150XFAM-LEED-FMKDMSOstocksolutionbyadding50μLofDMSOtothevialofFAM-LEED-FMK(ComponentA). Add150XFAM-LEED-FMKintothecellsolutionata1:150ratio,andincubatethecellsina37°C,5%CO2incubatorfor1hour.
Note1:Thecellscanbeconcentratedupto~5X106cells/mLforFAM-LEED-FMKlabeling.Theunused150XFAM-LEED-FMKDMSOstocksolutionshouldbedividedassingleusealiquotandstoredat-20°C.
Note2:Foradherentcells,gentlyliftthecellswith0.5mMEDTAtokeepthecellsintact,andwashthecellsoncewithserum-containingmediapriortoincubationwithFAM-LEED-FMK.
Note3:Theappropriateincubationtimedependsontheindividualcelltypeandcellconcentrationused.Optimizetheincubationtimeforeachexperiment.
3. Spindownthecellsat~200gfor5minutes,andwashcellswith1mLwashbuffer(ComponentB)twice.Resuspendthecellsindesiredamountofwashingbuffer.
Note1:FAM-LEED-FMKisfluorescent,thusitisimportanttowashoutanyunboundreagenttoeliminatethebackground.
Note2:Fordetachedcells,theconcentrationofcellsshouldbeadjustedto2-5X105cells/100μLaliquotpermicrotiterplatewellforuseinstep5.
4. Ifdesired,labelthecellswithaDNAstain(suchaspropidiumiodidefordeadcells,orHoechstforwholepopulationofthecellnucleusstain).
5. Monitorthefluorescenceintensitybyfluorescencemicroscopy,flowcytometer,orfluorescentmicroplatereaderatEx/Em=490/525nm(forpropidiumiodide,Ex/Em=535/635nm;forHoechstdyes,Ex/Em=350/461nm).
5.1 Forflowcytometry,monitorthefluorescenceintensityusingtheFL1channel(FL2channelforpropidiumiodidestaining).Gateonthecellsofinterest,excludingdebris.
5.2 Forfluorescencemicroscopyandfluorescentmicroplatereader.Place100μLofthecellsuspensionsintoeachofwellsofa96-wellblackmicrotiterplate.
Note: Ifitisnecessarytoequilibratethecellconcentrations,adjustthesuspensionvolumefortheinducedcellstoapproximatethecelldensityofthenon-inducedpopulation. Thisadjustmentstepisoptionalifyourcelltreatmentdoesnotresultinadramaticlossinstimulatedcellpopulationnumbers.
5.3 ObservecellsunderafluorescencemicroscopeusingFITCchannel(TRITCchannelforpropidiumiodidestaining,DAPIchannelforHoechststaining).
5.4 MonitorthefluorescenceintensityusingEx/Em=490/525nm(cutoffat515nm)bottomreadmodeusingafluorescentmicroplatereader.
| References&Citations |  CitationExplorer |
HelicobacterpyloriSecretedProteinHP1286TriggersApoptosisinMacrophagesviaTNF-IndependentandERKMAPK-DependentPathways
Authors:RaquelTavares,SushilKumarPathak
Journal:FrontiersinCellularandInfectionMicroBIOLOGy(2017):58
Deathreceptor3mediatesnecroptoticcelldeath
Authors:SebastianBittner,GertrudKnoll,MartinEhrenschwender
Journal:CellularandMolecularLifeSciences(2016):1--12
HelicobacterpyloriproteinJHP0290exhibitsproliferativeandanti-apoptoticeffectsingastricepithelialcells
Authors:RaquelTavares,SushilKumarPathak
Journal:PloSone(2015):e0124407
