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:
- Specificallymeasureepithelialapoptosisincellcultureapplications
- Theassayhasadynamicrangeandsensitivitysuitableforinvitrowork
- SpecificquantificationmeasurementtoolforapoptosisinCK18positivecells
- Measuresastable,accumulatedproductallowingformeasurementat1latertimepoint
- SandwichELISAina96wellplateinaconvenientready-to-useformat
- Easytoperform,onlyaminimumofpipettingstepsrequired
- CK18ispresentinsimple epithelialcellsonly,thusincreasingspecificityofmeasurementinepithelial-basedmodels
- Powerfultoolforinvitrocharacterizationofapoptosis-inducingdrugs.
- Mesauresformationofacapase-cleavageproductintheinteriorofspheroids
- Useoftissueslicesfromexvivotumorstomeasureapoptosisofcandidatedrugs
- Keyadvantageinco‐culturesystemstodeterminedrugeffectonepithelialtissue
CK18KitComposition:
Reagents,Packaging,Storage,andStABIlity
- M6CoatedMicrostrips: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).
