HO-1 and bilirubin protect ECs against complement-mediated injury. HUVECs were treated for 24 hours with bilirubin (10 μM; ) or vehicle alone (UT; ■) before harvesting, opsonization with mAb RMAC8, and exposure to NHS or heat-inactivated NHS (HIHS) for up to 3 hours. To inhibit DAF, mAb 1H4 was added to the assay with RMAC8. (A) C3 deposition was analyzed by flow cytometry using FITC-conjugated anti–human C3, and results are RFI (mean ± SEM; n = 6 experiments). *P < .05. (B) To measure complement-mediated lysis, propidium iodide (PI; 50 μg/mL) was added to the cell suspension and analysis was by flow cytometry. Percentage EC lysis was calculated as the number of PI-positive cells expressed as a percentage of the total number of cells (mean ± SEM; n = 6 experiments). *P < .05. (C) Murine cardiac ECs isolated from Hmox-1^−/− and Hmox-1^+/+ mice were opsonized with antiendoglin mAb MJ7/18 and incubated with 5% to 10% normal mouse serum (NMS) or heat-inactivated NMS (HIMS) for 90 minutes at 37°C, before analysis of C3 deposition by flow cytometry using FITC-labeled antimouse C3. Results are RFI (mean ± SEM; n = 3 experiments). *P < .05. (D) To estimate complement-mediated cell lysis, murine ECs were opsonized with MJ7/18 and incubated with NMS or HIMS, respectively, for 2 hours at 37°C. After washing, ECs were resuspended in veronal-buffered saline and propidium iodide added (50 μg/mL). ECs were analyzed by flow cytometry and percentage EC lysis calculated as in panel B (mean ± SEM; n = 3). *P < .05.