C3 gene-silenced macrophages have lowered ability to stimulate alloreactive T cells. Thioglycollate-elicited C3^+/+ macrophages were transfected with 100 nM of either C3 siRNA or control siRNA using a siRNA transfection kit (Qiagen). After 48 hours of transfection, C3 expression was measured by quantitative real-time RT-PCR (A) and ELISA (B). After 48-hour transfection, 1 × 10⁵ macrophages were cocultured with 1 × 10⁵ primed alloreactive CD3⁺ T cells for 2 days. Supernatants were collected and used for measuring the production of IL-2 by ELISA (C). All data were generated from 3 independent experiments, with macrophages pooled from 5 mice in each experiment and analyzed by Student t test. P values are for comparisons between untreated C3^+/+ macrophages and other groups of macrophages, ^***P < .001; ns, no significant difference. Error bars indicate SEM.