Extracellular C3 does not account for the different ability of C3^-/-and C3^+/+macrophages to stimulate alloreactive T cells. Thioglycollate-elicited macrophages were prepared from C3^+/+ and C3^-/- mice (n = 5/group). (A) C3^+/+ or C3^-/- macrophages and C3^+/+ macrophages that were preincubated with 25% mouse serum at 37°C for 30 minutes and washed 3 times were stained for deposition of C3 using FITC-conjugated goat anti-mouse C3 IgG F(ab′)²  fragment. In all histogram plots, the control peak (dashed line) corresponds to unstained cells. The detection peak (solid line) shows the binding of C3 antibody. (B) C3^+/+ or C3^-/- macrophages were cocultured with primed alloreactive CD4⁺ T cells in the absence or presence of either WT (wild-type) normal serum or C3^-/- serum for 5 days. Supernatants were collected and used for measuring the production of IFN-γ by ELISA. Data were analyzed by Student t test and showed no significant difference between untreated C3^-/- macrophages and serum-treated C3^-/- macrophages. Error bars indicate SEM. Results presented in panels A and B are representative of 3 independent experiments.