Complement-Dependent Cytotoxicity (CDC) Assay

The CDC assays were carried out using WIL2-S cells as target cells and complement derived from human serum (Quidel Corporation). Etrolizumab and the anti-CD20 antibody rituximab were serially diluted in assay medium (RPMI-1640 supplemented with 20 mM HEPES pH 7.2, 0.1% BSA, and 0.1 mg/mL gentamicin), and distributed into a 96-well tissue culture plate (Costar; Corning Inc.). Following the addition of human serum complement (diluted 1:3 in assay medium) and the target cells (10⁵ cells/well), the plate was incubated 12 h at 37°C. After the incubation, AlamarBlue was added at 50 μL/well and the plate was incubated for an additional 15–18 h. The CDC of the test antibody was quantified through absorbance at 530 nm excitation with 590 nm emission on a fluorescence plate reader (SpectraMax GeminiXS, Molecular Devices). The EC50 values were generated by fitting the data to a four-parameter equation (SoftMax Pro).