C5b-9 deposition on PIPLC-treated human EA.hy926 cells. (A) Flow cytometric analysis of CD59 expression on ADP-activated EA.hy926 l cells treated with PIPLC vs untreated EA.hy926 cells. After trypsinization, endothelial cells were recovered in cell culture medium at 37°C for 30 minutes, treated with PIPLC, and then stained with anti-CD59 antibody. Reduced CD59 expression on PIPLC-treated cells is shown. (B-E) Confocal microscopy of ADP-activated EA.hy926 cells treated with PIPLC and stained with C5b-9 (depicted in red) and 4,6 diamidino-2-phenylindole (blue) as nuclei cell counterstaining. Magnification 40x. C5b-9 deposition is shown after incubation with aHUS serum (B) compared with heat-inactivated aHUS serum (C), TTP serum (D) and normal serum (E). PIPLC: Phosphatidylinositol-specific phospholipase C; aHUS: atypical hemolytic uremic syndrome; Heat-aHUS: heat inactivated atypical hemolytic uremic syndrome; TTP: thrombotic thrombocytopenic purpura.