SPIO-mediated complement activation in defined human sera. (a) Effect of SPIO NW and dextran concentration on complement activation, based on soluble C5b-9, in a typical human serum from a healthy Caucasian individual. SPIO NW concentration represents an equivalent dextran concentration in the preparation. Zymosan (1 mg/ml) was used as positive control for monitoring complement activation; (b) contribution of Ca2+-sensitive pathways and the AP turnover to dextran- and SPIO-mediated complement activation in the same serum as panel (a). The background level with EGTA alone is similar to EGTA + Mg2+ (data not shown); (c) dextran and SPIO NW both enhanced the AP turnover as shown in panel as monitored by Bb generation; (d) SPIO-mediated complement generation of C4d is independent of C1q suggesting involvement of the LP; (e) MBL is required for SPIO-mediated triggering of the LP. The final concentration of iron in panels (b-e) was 200 μg/mL (equivalent dextran concentration). C1qdepl and MBLdef represent sera immunochemically depleted from C1q and genetically deficient from MBL, respectively. MBL and L-ficolin concentrations are shown in for C1qdepl and MBLdef sera. We used serum from a human donor with high titer of MBL to purify MBL/MASP complexes. The same serum was used to deplete C1q. In experiments where we added MBL/MASP to a serum genetically deficient in MBL, the final equivalent concentration of MBL was 1330 ng/mL (to resemble average MBL concentration in human serum) *p < 0.05, and **p < 0.01.