Neutrophils activate the complement AP and vice-versa. (A) Anti-C3d MFI of isolated PMNs labeled after a 30-minute incubation (2 × 10⁶/mL) at 37°C in HBSS²⁺ in BSA-coated tubes, without (0) or with 10 ng/mL TNF-α ± 10⁻⁶M fMLP for the last 15 minutes of incubation, or with 10 ng/mL PMA, followed by a 30-minute incubation with one-third vol/vol autologous lepirudin-plasma (mean ± SD, n = 6). (Inset) Fluorescence histograms of TNF/fMLP activated PMNs labeled with anti-C3c, anti-C3d, and anti-iC3b mAbs or with the IgG1 control (shaded peak). (B-D) Anti-C3d (B-C) and anti-CD11b (D-E) MFI, measured by flow cytometry, on PMNs preincubated in HBSS²⁺ (buffer), 30 minutes with TNF-α 2 ng/mL (TNF), or TNF/fMLP (TNF/fMLP) as defined in “Results,” followed by a 30-minute incubation with one-third AB-serum without (NHS) or with 10mM EDTA (NHS-EDTA), with one-third factor B-depleted serum without (B⁻) or with added 100 μg/mL purified factor B (B⁻ + B) or with one-third C2-depleted (C2⁻) serum (mean ± SD, n = 4 for B, D; n = 3 for C, E). Statistical analysis comparisons were performed between samples with B⁻ serum and the corresponding NHS control. *P < .05. **P < .01. ***P < .001. (F) Oxidative response of PMNs pretreated for 10 minutes with 5μM DCFHDA before stimulation by TNF-α and incubation in one-third normal or depleted sera. DCFHDA index of PMNx submitted to stimulus x in serum x = (MFIx − MFI resting PMNs in NHS) × 100/MFI x-stimulated PMN in NHS − MFI resting PMN in NHS).