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Ex/Em(nm)	492/514
MW	N/A
CAS#	N/A
Solvent	Water
Storage	F/D/L
Category	CellAnalysis CellApoptosis
Related	ApoptosisandCytotoxicity BiochemicalAssays

OurCellMeter™livecellcaspasesactivityassaykitsarebasedonfluorescentFMKinhibitorsofcaspases.Theseinhibitorsarecellpermeableandnon-cytotoxic.Onceinsidethecell,thecaspaseinhibitorsbindcovalentlytotheactivecaspases.ThisCellMeter™LiveCellCaspase13ActivityAssayKitisdesignedtodetectcellapoptosisbymeasuringcaspase13activationinlivecells.Itisusedforthequantificationofactivatedcaspase13activitiesinapoptoticcells,orforscreeningcaspase13inhibitors.FAM-LEED-FMK,thegreenlabelreagent,allowsfordirectdetectionofactivatedcaspase13inapoptoticcellsbyfluorescencemicroscopy,flowcytometer,orfluorescentmicroplatereader.Thekitprovidesalltheessentialcomponentswithanoptimizedassayprotocol.

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

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

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