Complement-dependent cytotoxicity (CDC) by cell counting kit-8 (CCK-8) assay

To show biological activity in the CD20 mAb (rituximab)—resistant (Ly10-R) OCI-Ly10 cell line, standard Cell Counting Kit-8 (CCK-8) assay were done to asses rituximab-mediated CDC. CD20 monoclonal antibody (rituximab)—sensitive (Ly10-S) or resistant (Ly10-R) OCI-Ly10 cells were centrifuged at 1200 rpm for 5 min, and then mixed with RPMI-10 in combination with human serum. Cell density was adjusted to 5 × 10³ cells per well and inoculated in 96 well cell culture plate with rituximab. Rituximab was added according to the above different concentrations (0, 16, 32, 64, 128 μg/ml), and three parallel microplate wells were set for each concentration. 10 µl CCK-8 reagent was added into each well at 37 °C and 5% CO₂ incubator for 2 h respectively, and the OD value of each well at 450 nm was measured by enzyme-linked immunosorbent assay after 4 h maintenance incubation. The inhibition rate was calculated by the following formula:growth inhibition rate = [OD value of the control g − roupOD value of the experimental group)/(OD value of the control group − OD value of the blank group) × 100%. All samples were run in triplicate in three different sets of experiments. Results are reported as a mean values with standard deviation (SD).