Complement activation on quiescent endothelial cells. Adherent, quiescent, and confluent HUVECs (Ai) or GEnCs (Bi) were incubated with 125 μL FB-depleted serum, supplemented with recombinant WT FB or supernatants, containing 4 μg mutant FB protein, to a final volume of 500 μL. C3-fragment deposition was assessed by flow cytometry with anti-C3c mouse monoclonal antibodies, followed by goat anti–mouse IgG-PE. Irrelevant mouse IgG1 was used as a control. The figure shows a representative histogram from 10 independent experiments with HUVECs and 3 with GEnCs. (B) Levels of sC5b-9 in the serum after incubation with HUVECs (i) or GEnCs (ii). Supernatants from 3 independent experiments, were tested by the Quidel ELISA kit sC5b-9 Plus. The level of sC5b-9 for the wild type in each experiment was taken as 100%. Results are presented as the mean of the independent experiments obtained for each mutation. The 2 mutations gave significantly higher C5b-9 formation compared with the WT (t test, P < .05). (C) Comparison between the levels of C3 deposition on quiescent, adherent GEnCs and HUVECs. The anti-C3c mean fluorescence intensity obtained with each mutant (each value being normalized with the isotype control) was presented as percentage of the WT. The fluorescence intensities for the WT and SN0 were comparable for HUVECs and GEnCs. Differences between mutants and WT, for each particular cell type, as well as differences between the 2 different cell types, for a given mutant, were statistically significant (n = 3, P < .05, t test). Potential artifacts due to endotoxin contamination were excluded by addition of endotoxin inhibitor polymyxin B or intentional contamination with LPS. These treatments did not change the levels of C3 deposition (data not shown).