Procedure 

1. Reagents
   • Heparin - 1000 U/ml
   • Ficoll-Hypaque
   • PBS
   • RPMI-1640 supplemented with 10 mM glutamine and 15% FBS
   • AET (0.14M) Dissolve 1.967 g AET in 35 ml di-H2O.
     Adjust to pH 8.0 with 1.0N NaOH. Bring volume to 50 ml with di-H2O.
     Store at 2-8oC. Check pH every 2 weeks.
2. AET-treated SRBC
   • Wash SRBC 4 times with PBS 
   • Add 4 volumes AET to 1 volume packed SRBC in a 15 m conical tube (1 ml of AET + 0.25 ml packed SRBC). 
   • Mix well. Incubate in a 37oC water bath for 30 minutes. Shake vigorously. 
   • Wash 3 times with PBS. 
   • Store in PBS at 2-8oC for up to 3 days.
3. SRBC-Absorbed FBS
   • Mix 10 volumes of FBS with 1 volume packed SRBC. 
   • Incubate at 37oC fir 30 minutes. 
   • Incubate at 2-8oC for 30 minutes. 
   • Centrifuge at 400 g for 10 minutes. 
   • Collect the FBS. Filter sterilize. Store aliquots at -20oC.
4. Preparation of PBL's
   • Draw peripheral blood into syringe containing 10 U/ml heparin.
   • Dilute the blood 1:1 with PBS.
   • Layer 30 ml of diluted blood onto 20 ml Ficoll-Hypaque. 
   • Centrifuge at 1550 rpm for 30 minutes, room temperature. 
   • Aspirate and discard the supernatant. 
   • Carefully collect the interface of PBL's and transfer into a clean tube. 
   • Fill the tube with PBS. Centrifuge at 1550 rpm for 10 minutes. 
   • Wash the pellet 2 times with PBS. 
   • Count the cells and resuspend to 10⁷ cells/ml in PBS.
5. Separation of T-Cells
   • Mix 1 ml of AET-treated SRBC with 10 ml FBS.
   • Mix and equal volume of PBL's with a 1% (v/v) mixture of AET-SRBC_FBS in a 50 ml tube.
   • Incubate in a 37oC water bath for 10 minutes. 
   • Centrifuge at 200 g for 10 minutes. Make sure that the cells have pelleted. If not, re-centrifuge for 5 minutes. 
   • Place the tube upright on ice for 60 min. 
   • Layer super over 15 ml of Ficoll-Hypaque leaving 7.5 ml of fluid above the pellet. 
   • Resuspend the pellet by rotating the tube along the long axis. 
   • Stand upright for 1 minute. Remove the top 5 ml and layer on Ficoll-Hypaque. 
   • Rotate as above and transfer to gradient tube. 
   • Wash the tube with 5 ml of PBS and add to gradient. 
   • Centrifuge at 300 g for 40 minutes, room temperature. 
   • Collect the B cells at the interface. Wash 3 times with PBS. 
   • Suspend the SRBC-T cell pellet. Centrifuge at 300 d for 10 minutes. 
   • Aspirate all of the supe. Break up the cell pellet by gently shaking. 
   • Add 9 ml of di-H2O. with shaking for 4 seconds. 
   • Add 1 ml of 10X PBS with shaking. 
   • Immediately fill the tube with 1X PBS. 
   • Centrifuge at 300 g for 10 minutes, and wash 2 times with PBS.