Support Protocols

Receptor-Mediated Apoptosis in Human Cells Using anti-Fas/CD95 (Clone DX2)

Materials Required

1. A cell line (eg, Jurkat T cells, ATCC TIB 152) or primary cells that can be induced to undergo apoptosis by human Fas mAb.
2. Anti-human Fas mAb, Clone DX2 (Cat. No. 555670).
3. Recombinant Protein G (SIGMA, Cat. No. P4689). The addition of Protein G to the tissue culture medium can significantly enhance the efficiency of DX2 mAb to induce apoptosis, presumably by cross-linking.
4. Tissue culture flasks or tissue culture 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.

Protocol

1. Induction of apoptosis: treat cell suspension (0.5-1 x 10⁶ cells/ml) with anti-CD95, clone DX2 (titrate 2-20 µg/ml to determine optimal concentration), and 2 µg/ml Protein G. Negative controls should consist of cells with medium alone (no mAb or Protein G) and cells with medium and Protein G (no mAb).
2. Perform a time course to obtain optimum results. An incubation time of 2-16 hr at 37°C is suggested.
3. Proceed with assays designed to evaluate induction of apoptosis.