Resistance to Fas-induced apoptosis of human TEC on day 4 of primary culture can be raised by concomitant addition of cycloheximide. TEC were collected on day 3 of primary culture and subcultured in 24-well plates. After a 24-hour period to allow cells to adhere, 0.5 μg/mL agonistic anti-Fas IgM antibody (clone CH-11) or 0.5 μg/mL mouse IgM antibody was added in the presence or absence of 10 μg/mL cycloheximide. (A) After cell fixation in ethanol containing 5% acetic acid, cells were stained with Toluidine blue and photographed. In the absence of cycloheximide, no change in cell density was observed (data not shown). When cycloheximide was added, cell density was clearly reduced in the presence of agonistic anti-Fas antibody (clone CH-11) in comparison to cells treated with IgM. (B) Living cells recovered from 0.5 × 106 cells subcultured in 24 wells on day 3 were counted after the culture by using the Trypan blue exclusion method. In the absence of cycloheximide, the number of cells collected was not significantly modified by anti-Fas. By contrast, when cycloheximide was added, the number of cells recovered was clearly reduced in the presence of anti-Fas. Data are the means ± SEM of three independent experiments. (C) Apoptosis was analyzed by quantifying phosphatidylserine residues exposed on the external cell membrane. Annexin-V binding was performed as previously described. A representative analysis shows that TEC undergo anti-Fas (CH-11)–induced apoptosis in the presence of cycloheximide, because the proportion of annexin-V–positive cells is increased relative to IgM treatment, whereas TEC were resistant in the absence of cycloheximide. (D) Data are the means ± SEM of four independent experiments.
