Expression of FasL in B lymphocytes before and after stimulation via HLA class II. (a) PMA and ionomycin; (b) bacterial superantigen TSST-1; (c) anti–HLA-DR MoAb L227 XL; (d) anti–HLA class I MoAb W6.32 XL. The level of fluorescence in nonstimulated cells is shown as an unfilled profile and is compared with the filled profile of stimulated cells. Profiles e to h show binding of an isotype-matched control antibody (polyclonal rabbit IgG, anti–cyclin B) before and after stimulation as above. For a to h, the cells were fixed in a solution containing 95% ethanol with 5% acetic acid and then stained with either 2 μg/mL anti-FasL antibody N-20 or 1 μg/mL anti-FasL PE62, followed by 10 μg/mL secondary MoAb conjugated with FITC. (i) Immunoblot with the anti-FasL antibody PE62 in which cell lysates were prepared either under nonreducing conditions (lanes 1, 2, and 5) or under reducing conditions (lanes 3, 4, and 6). Lanes 1 and 3, nonstimulated cells; lanes 2 and 4, L227 XL; lanes 5 and 6, bacterial superantigen TSST-1.