Complement dependent cytolysis (CDC) and antibody dependent cell-mediated cytotoxicity (ADCC)

Calcein AM release (Molecular Probe) was used for measuring cytolysis (32). For CDC, cells (2 × 10⁴ cells/ml) were seeded in a 96-well plate and grown to confluency. Cells were incubated with calcein AM (5 μg/ml) for 30 min at 37°C, washed twice prior to treatment with anti-c-Kit mouse IgG2a monoclonal antibody (Thermo scientific) or anti-GPC3 mouse IgG2a antibody (Santa Cruz, CA) together with anti-CD55 mouse IgG1 (BRIC216, EMD Millipore, MA), anti-CD46 (GB24, kindly provided by Dr. John Atkinson, Washington University, St. Louis) or anti-CD59 (YTH53.1, Santa Cruz, CA) for 20 min at 37°C. Cells were incubated in the presence of normal human serum (NHS) as a source of complement at 37°C for 4h. Alternatively, BHK21 cells were infected with vesicular stomatitis virus (VSV, Indiana) at a multiplicity of infection of 0.3. Cells were incubated for 16h before addition of calcein AM. VSV G antibody (SantaCruz, CA) and a 5% dilution of normal human serum in the presence or absence of purified CD55 protein (ACRO Biosystem, Newark, DE), or each conditioned medium. The supernatant was transferred to a new 96 well plate to represent the complement-mediated release of calcein AM fraction. The calcein AM remaining in the cells were released by incubation with 0.1% Triton X-100 for 30 min as maximum release. Samples were measured using a Multimode Plate Reader using an excitation filter 485 nm and emission filter 530 nm (PerkinElmer). Percent lysis was calculated according to the formula [(test release − spontaneous release)/(maximum release − spontaneous release)] × 100. For ADCC, cells stained by calcein AM were treated with each antibody at 37°C for 20 min. Cells were incubated with NK 3.3 effector cells at 37°C for 4h. The supernatant was transferred and the fluorescence was measured similar to CDC assay. Percent lysis was calculated using the same formula with CDC.