SUPPORT PROTOCOLS

Receptor-Mediated Apoptosis in Mouse Cells Using anti-Fas/CD95 (Clone Jo2)

Materials Required

  1. A cell line or primary cells that can be induced to undergo apoptosis by mouse Fas mAb. We typically use thymocytes isolated from a 4-6 week old BALB/c mouse.
    Note: Not all cell types that express Fas can be induced to undergo apoptosis using this protocol.
  2. Anti-mouse Fas mAb, Clone Jo2 (Cat. No. 554254).
  3. Recombinant Protein G (SIGMA, Cat. No. P4689). The addition of Protein G to the tissue culture medium can enhance the efficiency of Jo2 mAb to induce apoptosis in some experimental systems.
  4. Tissue culture flasks or plates.
  5. RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine and 1% antibiotics (penicillin/streptomycin; 100 U/ml). This supplemented medium is referred to as 'medium' below.

Procedure

  1. Isolate BALB/c thymocytes from the thymus of a 4-6 week old mouse.
  2. Treat cell suspension (2 x 106-1 x 107 thymocytes/ml) with 2-20 µg/ml Jo2 mAb. Negative controls should consist of cells cultured in medium without the addition of anti-Fas and medium containing a hamster isotype control (clone Ha4/8, Cat. No. 553961) at the same concentration as the anti-Fas mAb. If Protein G (1-2 µg/ml) is added along with Jo2, then add controls containing (1) Protein G alone and (2) Protein G plus the Ha4/8 mAb.
  3. Perform a time course to obtain optimum results. Incubation time between 2 to 12 hr at 37°C is suggested.
  4. Proceed with assays designed to evaluate induction of apoptosis.