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T and B cell Isolation

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/**/

Author:NanciEDonacki

Source:ContributedbyNanciEDonacki

Abstract:IsolateTandBcellfromperipheralblood.

Procedure

Reagents
- Heparin-1000U/ml
- Ficoll-Hypaque
- PBS
- RPMI-1640supplementedwith10mMglutamineand15%FBS
- AET(0.14M)Dissolve1.967gAETin35mldi-H2O.AdjusttopH8.0with1.0NNaOH.Bringvolumeto50mlwithdi-H2O.Storeat2-8oC.CheckpHevery2weeks.
AET-treatedSRBC
- WashSRBC4timeswithPBS
- Add4volumesAETto1volumepackedSRBCina15mconicaltube(1mlofAET+0.25mlpackedSRBC).
- Mixwell.Incubateina37oCwaterbathfor30minutes.Shakevigorously.
- Wash3timeswithPBS.
- StoreinPBSat2-8oCforupto3days.
SRBC-AbsorbedFBS
- Mix10volumesofFBSwith1volumepackedSRBC.
- Incubateat37oCfir30minutes.
- Incubateat2-8oCfor30minutes.
- Centrifugeat400gfor10minutes.
- CollecttheFBS.Filtersterilize.Storealiquotsat-20oC.
PreparationofPBL"s
- Drawperipheralbloodintosyringecontaining10U/mlheparin.
- Dilutetheblood1:1withPBS.
- Layer30mlofdilutedbloodonto20mlFicoll-Hypaque.
- Centrifugeat1550rpmfor30minutes,roomtemperature.
- Aspirateanddiscardthesupernatant.
- CarefullycollecttheinterfaceofPBL"sandtransferintoacleantube.
- FillthetubewithPBS.Centrifugeat1550rpmfor10minutes.
- Washthepellet2timeswithPBS.
- CountthecellsandresUSPendto10⁷cells/mlinPBS.
SeparationofT-Cells
- Mix1mlofAET-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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T&nbsp;and&nbsp;B&nbsp;cell&nbsp;Isolation

T and B cell Isolation