Inhibition of RCAS1-induced apoptosis by anti-Fas neutralizing antibody and Fas-Fc
| Culture additives . | Experiment 1 . | Experiment 2 . | ||
|---|---|---|---|---|
| (−) . | + 4B4-3B . | (−) . | + Fas-Fc . | |
| Control | 7.6 ± 0.4 | 6.8 ± 0.1 | 15.4 ± 3.1 | 17.2 ± 0.8 |
| + CH-11 | 47.6 ± 1.4 | 9.1 ± 0.8* | 26.9 ± 1.9 | 16.3 ± 0.9† |
| + nRCAS1 | 40.9 ± 1.1 | 42.8 ± 2.0 | 26.7 ± 2.4 | 24.2 ± 0.9 |
| Culture additives . | Experiment 1 . | Experiment 2 . | ||
|---|---|---|---|---|
| (−) . | + 4B4-3B . | (−) . | + Fas-Fc . | |
| Control | 7.6 ± 0.4 | 6.8 ± 0.1 | 15.4 ± 3.1 | 17.2 ± 0.8 |
| + CH-11 | 47.6 ± 1.4 | 9.1 ± 0.8* | 26.9 ± 1.9 | 16.3 ± 0.9† |
| + nRCAS1 | 40.9 ± 1.1 | 42.8 ± 2.0 | 26.7 ± 2.4 | 24.2 ± 0.9 |
Two experiments to inhibit the stimulation of Fas were done on day 7 ECFCs. Experiment 1: Anti-Fas neutralizing MoAb (clone 4B4-3B, 1.0 μg/mL) was added to the liquid cultures, which were incubated for 1 hour at 37°C. After this, Fas-stimulating antibody (0.5 μg/mL; clone CH-11) or nRCAS1 (40 μg/mL) was added and incubation was carried out for a further 24 hours. Experiment 2: Fas-Fc chimeric protein, which reduces Fas-mediated apoptosis by occupying FasL (100 ng/mL), was preincubated with CH-11 or nRCAS1 for 1 hour at 37°C and were then added to the culture media. In both experiments, apoptotic cells were detected by annexin V binding assay. Values are mean ± SD from triplicate determinations.
nRCAS1 indicates a soluble form of RCAS1; MoAb, monoclonal antibody. See Table 1 for other abbreviations.
Versus without 4B4-3B, P < .001.
Versus without Fas-Fc, P < .001.