Complement-mediated Cytotoxicity Assay

CP convertase assay was performed as described in [17 (link)], with further modifications described in [18 (link)]. Briefly, cells were loaded with calcein AM, then pelleted in a V-shaped plate, overlaid with 25 µL of PBS, and incubated at 37 °C with shaking at 600 rpm. A mixture of ofatumumab (100 µg/mL) and 20% of C5-depleted serum (Complement Technology) (Tyler, TX, USA) supplemented with 5 µg/mL of respective C2 variant (all in a total volume of 25 µL) were added at a specific time point. Then, cells were washed with EDTA-GVB buffer (40 mM EDTA, 5 mM veronal buffer, 0.1% gelatin, 145 mM NaCl), pelleted, and overlaid with EDTA-GVB buffer containing 1:20 dilution of guinea pig serum (Harlan Laboratories) (Indianapolis, IN, USA). The readout of calcein release was performed as in the CDC assay.