Complement-dependent cytotoxicity assay and antibody-dependent cellular cytotoxicity

Macrophages were purified from the peritoneal cavity flushing fluid of BALB/c mice using CD14 microbeads. Target cells, which were the colon cancer cell lines HCT116 and CT26, were plated to 96-well plates (1×10⁵ in 100 μl per well). Then, infliximab (10 μg/ml) and/or guinea pig serum with active complements was added for incubation for 5 h at 37°C with 5% CO₂ to conduct the complement-dependent cytotoxicity (CDC) assay. For antibody-dependent cellular cytotoxicity (ADCC) assay, after 1 h of Oxa treatment, the effector cells (macrophages) were added for incubating for 48 h at 37°C with 5% CO₂. The cells were then subjected to cell viability assay. The cell cytotoxicity was calculated by following the method described by Yu [22].