C3-convertase formation from purified AP components, on the surface of neutrophils. PMNs (2 × 10⁶/mL in HBSS²⁺) in BSA-coated tubes were either kept in buffer (resting) or activated for 30 minutes with 10 ng/mL TNF-α at 37°C (TNF) and a sample used to measure cell-bound properdin by flow cytometry (upper panel). PMNs were then washed with HBSS⁻ and resuspended at 2 × 10⁶/mL in HBSS, with a final 2mM concentration of Ca²⁺ and Mg²⁺, containing 330 μg/mL pure C3 either alone (C3, thin line) or with factor B 100 μg/mL and factor D 1 μg/mL (C3,B,D dark line) or with factors B and D and properdin 2 μg/mL (C3,B,D,P dotted line), for 30 minutes at 37°C. The negative control was given by a sample of PMNs incubated for 30 minutes in one-third NHS-ethylenediaminetetraacetic acid (gray peak). Cells were then washed and cell-bound C3 measured by flow cytometry. The right shifts from the gray peak background position represent the binding of C3 to neutrophils in each sample. Flow cytometry histograms of one representative experiment, of 3 similar ones, are shown.