Functional analysis of cytokine-induced DAF on ECs. HMECs were plated at confluence in 35-mm petri dishes (6 × 10⁵ cells/dish) and cultured overnight at 37°C. They were then stimulated with a combination of TNF- (10 ng/mL) and IFN-γ (500 U/mL) for 48 hours. After harvesting, ECs were incubated with the anti-endoglin MoAb RMAC8 or plain medium alone for 30 minutes at 4°C. In the blocking experiments, the anti-DAF MoAb 1H4, anti-CD59 MoAb A35, or isotype-matched negative control MoAb (final concentration, 50 μg/mL) were added to the assay with RMAC8. The ECs were then washed in HBSS/1% BSA before addition of 20% NHS for 2 hours at 37°C. Binding of C3 was detected by flow cytometry using FITC-conjugated rabbit anti–human C3. (A) Percent C3 binding (mean ± SD) to unstimulated (▪) and TNF- and IFN-γ–stimulated (▧), HMECs, with binding to unstimulated ECs shown as 100%: (B) changes in C3 binding (RFI mean ± SD) on unstimulated (▪) and TNF- and IFN-γ–stimulated (▧) HMECs in the presence of non–complement-fixing inhibitory MoAbs 1H4 (anti-DAF) and A35 (anti-CD59). *P < .05.