Complement activation by leukocytes in whole blood. Samples (100 μL) of whole blood with lepirudin were incubated at 37°C without or with PMA 10 ng/mL for 30 minutes or with TNF-α 10 ng/mL for 15 minutes, followed by a 15-minute incubation with fMLP 10−6M. After several washes, they were labeled with anti-C3d (“Immunolabeling and flow cytometry”). (A) FACSScan FSC/SSC dot blot analysis of all leukocytes. (B-D) Anti-C3d fluorescence histograms of cells restricted to the neutrophil (B), monocyte (C), or lymphocyte (D) FSC/SSC gate, from unstimulated blood (bold line) or blood stimulated with TNF/fMLP or PMA (thin lines). The shaded peak represents the isotypic IgG1control labeling. (E) Anti-C3d MFI measured on neutrophils from blood drawn in 20mM EDTA 12.5mM EGTA, without or with 3mM Mg2+, or in lepirudin, activated or not with TNF 10 ng/mL 30 minutes, fMLP 10−6M 15 minutes, or TNF/fMLP as in panel B (mean ± SD, n = 3 experiments).
