Mouse B cell culture

B cell purification and activation assay

1. Collect Spleen from C57/BL6 mice and  place in PBS wash (PBS with 2% FBS, Pen/Strep, 5mM HEPES)
2. Harvest cells from spleen as usual  (Grind, ACK lysis, wash)
3. Resuspend cells in B cell culture media (RPMI 1640 with 10%FBS, + L-glutamine, Pen/Strep, bME (50µM), 5mM HEPES buffer) and count cells. Purify cells using EasySep mouse B cell isolation kit from StemCell Technologies. Should get 30-50 million purified B cells. 
4. Keep some cells to check for purity by flow cytometry.
5. For proliferation assays, label the remaining cells with CFSE. Resuspend cells in 500 µl of PBS and add 500 µl of 5 µM CFSE. (stock is 5 mM CFSE. Add 1 µl of CFSE to 1 ml of PBS). Pipette up and down.
6. 5 mins at RT (room temperature).
7. Quench with B cell media 5ml.
8. Spin down 5 mins @ 500 X g.
9. Stimulate cells with various mitogens such as:
   1.  LPS (10 µg/ml final)
   2. Anti-IgM (10 µg/ml, goat anti-mouse IgM Fab₂ fragment, Southern Biotech)+ recombinant mouse IL-4 10 ng/ml (R&D systems))
10. Add stimulants in 50 µl media / well in a 96 well flat bottom plate.
11. Make drugs in 1 ml of media. Resuspend the purified B cells ( 700,000 cells  in 350 µl of drug solution) : 100,000 per well.
12. Plate 50 µl each well (each condition in triplicate)
13. Plate controls as well (No stimulant, cells with no CFSE)
14. Culture cells for 64-72hr. Collect and stain with 7AAD or Annexin V to mark dead and dying cells.