Complement Classical Pathway Activity Assay

The complement haemolytic 50% activity (CH50) assay was performed to determine the function of the complement classical pathway as described previously (Li et al., 2013 (link)). Briefly, antibody‐sensitized chicken red blood cells (Colorado Serum Company, Denver, CO, cat#31151) were incubated for 30 min at 37°C with serial dilutions of rat serum samples in gelatin‐veronal buffer (GVB⁺⁺ buffer, Complement Technology, Tyler, TX, cat#B100). After centrifugation, the supernatant was transferred to a new plate, and the absorbance of the supernatant was determined at 405 nm by SpectraMax microplate reader (Molecular Devices, San Jose, CA). The fold serum dilution inducing 50% of complement haemolytic activity was determined and presented as the CH50 value.