ReagentsHeparin-1000U/mlFicoll-HypaquePBSRPMI-1640supplementedwith10mMglutamineand15%FBSAET(0.14M)Dissolve1.967gAETin35mldi-H2O.AdjusttopH8.0with1.0NNaOH.Bringvolumeto50mlwithdi-H2O.Storeat2-8oC.CheckpHevery2weeks.AET-treatedSRBCWashSRBC4timeswithPBSAdd4volumesAETto1volumepackedSRBCina15mconicaltube(1mlofAET+0.25mlpackedSRBC).Mixwell.Incubateina37oCwaterbathfor30minutes.Shakevigorously.Wash3timeswithPBS.StoreinPBSat2-8oCforupto3days.SRBC-AbsorbedFBSMix10volumesofFBSwith1volumepackedSRBC.Incubateat37oCfir30minutes.Incubateat2-8oCfor30minutes.Centrifugeat400gfor10minutes.CollecttheFBS.Filtersterilize.Storealiquotsat-20oC.PreparationofPBL"sDrawperipheralbloodintosyringecontaining10U/mlheparin.Dilutetheblood1:1withPBS.Layer30mlofdilutedbloodonto20mlFicoll-Hypaque.Centrifugeat1550rpmfor30minutes,roomtemperature.Aspirateanddiscardthesupernatant.CarefullycollecttheinterfaceofPBL"sandtransferintoacleantube.FillthetubewithPBS.Centrifugeat1550rpmfor10minutes.Washthepellet2timeswithPBS.CountthecellsandresUSPendto107cells/mlinPBS.SeparationofT-CellsMix1mlofAET-treatedSRBCwith10mlFBS.MixandequalvolumeofPBL"switha1%(v/v)mixtureofAET-SRBC_FBSina50mltube.Incubateina37oCwaterbathfor10minutes.Centrifugeat200gfor10minutes.Makesurethatthecellshavepelleted.Ifnot,re-centrifugefor5minutes.Placethetubeuprightonicefor60min.Layersuperover15mlofFicoll-Hypaqueleaving7.5mloffluidabovethepellet.Resuspendthepelletbyrotatingthetubealongthelongaxis.Standuprightfor1minute.Removethetop5mlandlayeronFicoll-Hypaque.RotateasaboveandtransfertogrADIenttube.Washthetubewith5mlofPBSandaddtogradient.Centrifugeat300gfor40minutes,roomtemperature.CollecttheBcellsattheinterface.Wash3timeswithPBS.SuspendtheSRBC-Tcellpellet.Centrifugeat300dfor10minutes.Aspirateallofthesupe.Breakupthecellpelletbygentlyshaking.Add9mlofdi-H2O.withshakingfor4seconds.Add1mlof10XPBSwithshaking.Immediatelyfillthetubewith1XPBS.Centrifugeat300gfor10minutes,andwash2timeswithPBS.

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