Introduction MatureTcellsrecognizeandrespondtotheantigen/MHCcomplexthroughtheirantigen-specificreceptors(TCR).ThemostimmediateconsequenceofTCRactivationistheinitiationofsignalingpathwaysincludinginductionofspecificproteintyrosinekinases(PTKs),breakdownofphosphatidylinositol4,5-biophosphate(PIP2),activationofproteinkinaseC(PKC)andelevationofintracellularcalciumionconcentration.Theseearlyeventsaretransmittedtothenucleusandresultin1.clonalexpansionofTcells,2.upregulationofactivationMarkersonthecellsurface,3.differentiationintoeffectorcells,4.inductionofcytotoxicityorcytokinesecretionand5.inductionofapoptosis.OneofthemostcommonwaystoassessTcellactivationistomeasureTcellproliferationuponinvitrostimulationofTcellsviaantigenoragoNISTicantibodiestoTCR.ThisprotocoliswrittenasastartingpointforexamininginvitroproliferationofmousesplenicT-cellsandhumanperipheralTcellsstimulatedviaCD3.Allstepswhereoptimizationbytheusercanbeachievedformeaningfulresultshavebeenhighlighted.Briefly,theseincludetitrationofthecellconcentrationused,titrationoftheactivatingreagentsandtheoptimalcultureperiodforthebestactivationresponse. Whatyouneed: Materials: -1XsterilePBS -Anti-mouseCD3e,145-2C11:FunctionalGrade,cat.no.16-0031orPurified,cat.no.14-0031 -SupplementedRPMI-1640<clickhere>forrecipe -Sterilesingle-cellsUSPensionofmousespleenorlymphnodes -96-wellflat-bottommicrotiterplateswithlids(Costarcat.no.3596) -MTTBuffer<clickhere>forrecipe -MTTLysingSolution<clickhere>forrecipe -ConcanavalinA,optional(ConA,Sigmacat.no.C5275) Instruments: -Pipettesandpipettors,Multichannelpipettor -Centrifuge -37degreesC,CO2incubator -96-wellmicrotestspectrophotometer ExperimentDuration: 20minutesantibodypreparationforcoatingwells 20minutespreparationofspleensinglecellsuspension 20minutestosetuptheassay 2-4daysincubation Method: ANTIBODYCOATINGOFTHEASSAYPLATE: 1)Preparea5-10ug/mlsolutionofanti-CD3e(145-2C11)insterilePBS.Calculatethenumberofwellsrequiredforeachexperimentalconditionandconsidertriplicatesamplesforeachcondition.Forexample,tocoatone-halfplate(48wells)2.6mlofantibodysolutionisrequired. Note:Wehaveperformedtitrationstudiesandfoundtheseconcentrationsof145-2C11toinduceamaximalresponse.However,apilotexperimenttodetermineefficacyofotherconcentrationsofthisantibodytoinducecellularactivationcanbeperformed.Forco-stimulationstudiesusingantibodiestootherantigens,asuboptimalactivationwithanti-CD3mayberequired.Toachievesuboptimalactivationviaanti-CD3,a0.5-0.1ug/ml145-2C11antibodysolutioncanbeused. 2)Dispense50uloftheantibodysolutionineachwellofthe96-wellassayplate.Forthecontrolunstimulatedwells,add50ulofsterilePBS. 3)TightlycovertheplatewithParafilmtoavoidsampleevaporationandincubateat37degreesCfor2hoursoralternativelypreparetheplateonedayinadvanceandkeepat4degreesCovernight. 4)Justbeforeaddingcells,removethe50ulantibodysolutionwithamultichannelpipettor. 5)Rinseeachwellwith200ulofsterilePBSanddiscardPBS. 6)Repeatstep5toremoveallunboundantibodyfromeachwell. 1)Harvestspleenandprepareasinglecellsuspensionundersterilecondition.Followtheredcelllysisprotocoltoremoveredcells.<clickhere>forRBClysisprotocol. 2)CountcellsandresuspendincompleteRPMI-1640at106/ml. Note:Thisconcentrationofspleencellsgivesagoodresponse.Ifexperimentalconditionsrequire,atitrationofcellconcentrations(2-3x106/mlto105/ml)shouldbeperformedforoptimization. 3)AfterwashingthewellswithPBS(step6above),add200ulofthecellsuspensiontoeachwellandplaceinahumidified37degreesC,5%CO2incubator. Note:Foranadditionalstimulationcontrol,incubatecellsin3wellswithConcanavalinAat1-4ugConApermlofculturemedium. 4)Incubatefor2-4days. Note:Proliferationofcellsbetweendays2and4givesagoodsignal;however,thisincubationtimecanalsobeoptimizedforspecificexperimentalconditions. 5)Add20uloftheMTTbuffertoeachwellandputbackintheincubatorfor4hours. 6)Add50uloftheMTTLysisSolutiontoeachwell,vortexgentlyandincubateovernight 7)Readtheplateat570nmthenextday. 8)Calculatethemeanandstandarderrorsandgraphthedata. ProtocolB:Stimulationofhumanperipheralbloodmononuclearcellswithanti-humanCD3monoclonalantibody;MTTassayfordetectionofcellularproliferation. HumanPBMCscanbeactivatedinvitrobysolubleanti-humanCD3antibodies.WehaveperformedtitrationstudieswiththeseantibodiesandestablishedthefollowingprotocolforstimulationofPBMC. Whatyouneed: Materials: -1XsterilePBS -Anti-humanCD3: OKT3,FunctionalGradecat.no.16-0037or HIT3a,FunctionalGradecat.no.16-0039 -SupplementedRPMI-1640<clickhere>forrecipe -SterilePBMC -96-wellflat-bottommicrotiterplateswithlids(Costarcat.no.3596) -MTTBuffer<clickhere>forrecipe -MTTLysingSolution<clickhere>forrecipe Instruments: -Pipettesandpipettors,Multichannelpipettor -Centrifuge -37degreesC,CO2incubator -96-wellmicrotestspectrophotometer ExperimentDuration: 30minutespreparationofPBMC 20minutestosetuptheassay 2-4daysincubation Method: 1)PreparePBMCandresuspendthecellsat1-2x106/mlofsupplementedRPMI. Note:ThisconcentrationofPBMCgivesagoodresponse.Ifexperimentalconditionsrequire,atitrationofcellconcentrations(2-3x106/mlto105/ml)shouldbeperformedforoptimization. 2)Add100ulofthecellsuspensiontoeachwell.Foreachcondition,usetriplicatewells. 3)Addsolubleantibodyin100ultoeachwell. 4)Placeinahumidified37degreesC,5%CO2incubator. 5)Incubatefor2-4days. Note:Proliferationofcellsbetweenday2and4givesagoodsignal;however,thisincubationtimecanalsobeoptimizedforspecificexperimentalconditions. 6)Add20uloftheMTTbuffertoeachwellandputbackintheincubatorfor4hours. 7)Add50uloftheMTTLysisSolutiontoeachwell,vortexgentlyandincubateovernight 8)Readtheplateat570nmthenextday. 9)Calculatethemeanandstandarderrorsandgraphthedata. Buffers&Media: SupplementedRPMI-1640: 900mlRPMI-1640(Hyclonecat.no.SH30027.02) 100mlFBS(Hyclonecat.no.SH30151.03)Heatinactivated(10%final) 1ml2-mercaptoethanol(GibcoBRLcat.no.21985-023) 10mlL-Glutamine(Hyclonecat.no.SH30034.01) Antibioticcocktail(optional) MTTBuffer: MTT(Sigmacat.no.M5655)makea5mg/mlstocksolutionin1XPBS-Keepprotectedfromlight. MTTLysingSolution: 20%SDS50%DMFProtocolA:StimulationofmouseperipheralTcellswithplate-bound145-2C11monoclonalantibody;MTTassayfordetectionofcellularproliferation.
ADDITIONOFCELLS
