Isolation of Tonsil B Cells

STEP 1 : Preparation of the sheep red blood cell suspension at 2%

Material required :

• Alsever solution (114 mM of dextrose, 27 mM of sodium citrate,71 mM of sodium chloride, 1 M of citric acid diluted in distilled water)
• Natural or synthetic sheep red blood cells.
• Phosphate Buffered Saline
• Neuraminidase 1 U/ml
• RPMI 1640 supplemented with 10% of heat-inactivated FBS, 2 mM of Glutamax, Penicillin (200 U/ml) and Streptomycin (100 µg/ml)

Protocol :

1. Incubate Neuraminidase at 37°C
2. Add 5 ml of Natural blood sheep or 2,5 ml of synthetic blood sheep to 5 ml of Alsever solution at room temperature (RT).
3. Incubated at RT for 5 minutes
4. Add PBS up to 50 ml and centrifuge at 900 g (or 2000 rpm) for 8 min at RT.
5. Remove supernatant and repeat step 3 until the supernatant are clear.
6. Add 1 ml of Neuraminidase 1U/ml to 1 ml of pellet (usually 4 ml).
7. Incubated at 37°C for 1 h. Mix frequently during the hour.
8. Add PBS up to 50 ml and centrifuge at 900 g (or 2000 rpm) for 8 min at RT.
9. Remove supernatant and repeat step 7 until the supernatant are clear (repeat at least 3 times to remove neuraminidase)
10. Resuspend the pellet up to 50 ml of cell culture media.
11. Store during 4 or 5 days at 4°C

STEP 2 : B cell isolation from tonsil

Material required :

• Tonsils (Fresh or stored in heat-inactivated FBS at 4°C during a maximum of 1 day)
• Petri dish
• PBS
• 2 Scalpels
• 2% Sheep red blood cells suspension previously prepared (stored at 4°C during a maximum of 5 days)
• 20ml Syringe
• Water bath at 37°C
• Pancoll human
• 40µm nylon cell stainer

Protocol :

1. Put tonsils in petri dish
2. Add 10ml of PBS at RT
3. Cut tonsil in tiny pieces with scalpels
4. Crush tonsil with a syringe plunger
5. Pipette off the supernatant and filter it with a 70µm cell stainer
6. Repeat 3 times step 2 to 5 to pipette off and filter all the supernatant
7. Dispatch filtered cell suspension on 2 x 10ml of Ficoll (1/3 of Ficoll, 2/3 of cell suspension)
8. Centrifuge at 650g for 30 min at RT, in swinging bucket rotor, without break nor acceleration
9. Aspirate the upper layer leaving the tonsil mononuclear cell (TMC) layer indisturbed at the interphase
10. Carefully transfer TMC to a new 50ml conical tube
11. Add PBS up to 40ml and centrifuge at 600g for 10min at RT
12. Remove supernatant and resuspend the pellet up to 40ml of PBS
13. Centrifuge at 300g for 10 min at RT
14. Resuspend the pellet with inactivated FBS and count cells
15. Add inactivated FBS up to a concentration of 10.10⁶ TMC/ml
16. Add 1ml of sheep red blood cells previously prepared for 10.10⁶ TMC
17. Mix carefully and incubate 10 min at 37°C
18. Mix carefully and deposit cell suspension on ficoll
19. Centrifuge at 650g for 30min at RT, in a swinging bucket rotor, without break nor acceleration
20. Aspirate the upper layer leaving the B cells at the interphase and transfer it in a new 50ml conical tube
21. Add PBS up to 40ml and centrifuge at 600g for 10min at RT
22. Optionnal: if the pellet contains too many red blood cells, perform ACK lysis before step 23 (resuspend the pellet in 4ml of ACK and put the tube 2 to 4 min in ice. Add PBS up to 50ml and centrifuge 300g for 10 min at RT)
23. Remove supernatant and resuspend the pellet of B cell up to 40ml of PBS
24. Centrifuge at 300g for 10min at RT
25. Check to purity of B cells and count cells.