Flow cytometric analysis of CRT on the cell surface 3.10^5 EL4 Thymoma cells, growing in suspension in RPMI 1640 (Gibco) supplemented medium were plated in 12-well plates and treated with mitomycin C (30mM, Sanofi Aventis) or cisplatin (25mM, Sigma) for 4h. Cells were harvested, washed once with cold PBS and possibly resuspended in 200mL of cold PBS containing 1mg of recombinant Calreticulin for 30 minutesutes on ice. After one wash with cold PBS, cells were fixed in 0.25% paraformaldehyde (PFA) in PBS for 5 minutesutes. After washing again once with cold PBS, cells were incubated for 30 minutes with primary antibody, diluted in cold blocking buffer (2% FBS in PBS), followed by washing and incubation with the Alexa488-conjugated monoclonal secondary antibody in blocking buffer (30 minutes). Each sample was then analyzed by FACScan (Becton Dickinson) to identify cell-surface Calreticulin. Secondary antibody alone was used as an isotype control, and the fluorescent intensity of stained cells was gated on propridium iodide (PI) negative cells.Pictures courtesy of Prof. Guido Kroemer, INSERM, Paris.