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.

ProtocolA:StimulationofmouseperipheralTcellswithplate-bound145-2C11monoclonalantibody;MTTassayfordetectionofcellularproliferation.

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.

ADDITIONOFCELLS

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.