Description:TheM30CytoDeath™ELISAisanovelresearchkit developedforcellcultureapplications.TheM30CytoDeath™assayhasadynamicrangeandsensitivitysuitableforinvitrowork.M30CytoDeath™ELISAisapowerfuldrugscreeningtool.Itisusefulforinvitrocharacterizationofapoptosis-inducingdrugs,includingestablishmentoftimecoursekineticsanddoseresponserelationships.TheeffectsoflargenumbersofsiRNAsonapoptosiscanbetestedinthe96wellformat.Theassayoffersauniquepossibilitytoquantifyapoptosisofepitheliallyderivedcellsinmulticellularspheroidsandorganculturesystems.TheM30CytoDeath™ELISAisbasedontheM30antibody(detectingtheAsp396caspase-cleavagesiteonCK18)andantibodyM6.ThiscombinationisdifferentfromthatusedintheM30-Apoptosense®ELISA.TheM30CytoDeath™ELISAisnotsuitableforserum/plasma.CK18KitAdvantages:SpecificallymeasureepithelialapoptosisincellcultureapplicationsTheassayhasadynamicrangeandsensitivitysuitableforinvitroworkSpecificquantificationmeasurementtoolforapoptosisinCK18positivecellsMeasuresastable,accumulatedproductallowingformeasurementat1latertimepointSandwichELISAina96wellplateinaconvenientready-to-useformatEasytoperform,onlyaminimumofpipettingstepsrequiredCK18ispresentinsimple epithelialcellsonly,thusincreasingspecificityofmeasurementinepithelial-basedmodelsPowerfultoolforinvitrocharacterizationofapoptosis-inducingdrugs.Mesauresformationofacapase-cleavageproductintheinteriorofspheroidsUseoftissueslicesfromexvivotumorstomeasureapoptosisofcandidatedrugsKeyadvantageinco‐culturesystemstodeterminedrugeffectonepithelialtissueCK18KitComposition:Reagents,Packaging,Storage,andStABIlityM6CoatedMicrostrips:Onemicroplate,12stripswith8wellseach,96dry wellsintotal.ThewellsarecoatedwithmousemonoclonalK18antibodyM6. Themicroplateissealedinanaluminiumbag,whichcontainsadesiccatingdevice. Ifnotallthestripsareused,resealthebagandkeepthedesiccatingdevice inside.Readyforuse!M30CytoDeathHRPConjugate:Concentrate(24×conc.).Onevialcontaining 0.4mLofmousemonoclonalM30antibody(anti-K18Asp396neo-epitope)conjugated withhorserADIshperoxidase(HRP)inaphosphatebufferwithprotein stabilizers.Preservativeadded.ShouldbedilutedwithM30CytoDeathConjugate DilutionBuffer(seeInstructionsforUsesection“ComponentPreparation”onpage9).Note!Do notexposetolight!M30CytoDeathConjugateDilutionBuffer:Onevialcontaining11mLofphosphate bufferwithproteinstabilizersfordilutionoftheM30CytoDeathHRPConjugate. Preservativeadded.Greencolored.M30CytoDeathStandards:StandardZerocontaining0.5mLofphosphate bufferwithfetalcalfserum(FCS).StandardsLow,MediumandHigh,0.5mL each,containingstandardmaterialinaphosphatebufferwithFCS.Thevaluesof theStandardsare0U/L(Zero),250U/L(Low),1000U/L(Medium)and3000U/L (High).Preservativeadded.Yellowcolored.Readyforuse!StandardZerocan beusedfordilutionsofsamples>3000U/L.WashTablet:Onetabletfor500mLofpreparedwashsolution.Dissolvethe WashTabletin500mLoffreshdeionisedwater.TMBSubstrate:Onebottlecontaining22mLofTMB(3,3’,5,5’-Tetramethylbenzidine) Substrate.Note!Donotexposetolight!Readyforuse!StopSolution:Onevialcontaining7mLof1.0Msulphuricacid.Readyforuse!SealingTape:One(1)sheet. MeasurementPrinciple:TheM30CytoDeath™ELISAisasolid-phasesandwichenzymeimmunoassay. StandardsandsamplesreactwithasolidphasecaptureantibodyM6directed againstK18andtheHRP(horseradishperoxidase)conjugatedM30antibody directedagainsttheK18Asp396neo-epitope.Unboundconjugateisremoved byawashingstep.TMBSubstrateisadded.Thecolordevelopmentisstoppedandtheabsorbanceisread.Theresultingcolorisdirectlyproportionaltothe concentrationoftheanalyte. Byplottingastandardcurvefromknownconcentrationsversusmeasuredabsorbance, theamountofantigeninthesamplecanbecalculated.Theconcentration oftheantigenisexpressedasunitsperliter(U/L).Background:Inastudy presentedattheSOTMeetingin2014,toxicitytestingoncryopreserveddifferentiatedHepaRG™cellswasperformedusingCK18kits.HepaRG™cellsexhibitmanycharacteristicsofprimaryhumanhepatocytes,includingmorphologyandexpressionofkeymetabolicenzymes,nuclearreceptors,anddrugtransporters.UnlikeHepG2cells,HepaRG™cellshavehighP450activityandcompleteexpressionofallnuclearreceptors.HepaRG™wasexposedtodifferentcompounds,includingparacetamol,chloropromazine,rotenone,rosiglitazoneandomeprazole.ToxicityandapoptosiswereassayedfromcollectedcellculturesupernatantsmeasuringM65andM30levels.M65resultswereconsistentwithcellviability,whereas,M30resultsindicatedthatapoptosiswasinducedatlowerdrugconcentrationswhilenecrosiswasmoreprominentathigherones(seefigurebelow).HumanHepRG™hepatocytestreatedwithdifferentcompounds.Toxicityandapoptosiswereassessedfromcollectedsupernatantsmeasuringfull-lengthK18(M65)andcaspase-cleavedK18(ccK18,M30)usingM65EpiDeath®andM30CytoDeath™ELISA.M65andM30kitsrobustlydetectcelltoxicityandapoptosisinHepaRGcells invitroexperiments. Aadditionalkeyfeatureisthatthe CK18kits measurean accumulated caspase-derivedproduct,sothereisnorisktomisstimeofapoptosisandtransientcaspaseactivation(Seefigurebelow).