Upregulation of DAF on ECs following assembly of the MAC of complement. HMECs were plated at confluence in 24-well plates (2 × 10⁵ cells/dish) and cultured overnight at 37°C. They were then opsonized with MoAb RMAC8 or negative control MoAb for 1 hour, followed by addition of 2.5% NHS to induce formation of the MAC. In parallel wells, ECs were pretreated with a combination of TNF- and IFN-γ for 16 hours before assembly of the MAC. Additional controls included the addition of HIHS or C7-deficient serum in place of NHS. To inhibit PKC, RO31-8220 was added for 30 minutes before assembly of the MAC and remained in the cultures throughout the assay. After a total incubation of 24 hours at 37°C, ECs were harvested and DAF expression was quantified by flow cytometry using an FITC-labeled anti-DAF MoAb.