Astrocyte Cytotoxicity Assay with AQP4-IgG and Complement

Astrocyte cultures were trypsinized, plated onto 96-well plates at 20,000 cells/well, and grown for 48 h. Human complement and AQP4-IgG were added in Hank’s balanced salt solution (HBSS, pH 7.2; Invitrogen), and cells were incubated at 37 °C for 2 h for cytotoxicity measurement by the Alamar Blue assay (Invitrogen) as described [25 (link)]. For C3d immunostaining, astrocyte cultures were exposed to AQP4-IgG and C6-depleted serum for 2 h, then washed and fixed with 4% paraformaldehyde (PFA). C6-depleted serum was used to visualize complement activation without cell lysis from membrane attack complex formation. After blocking, cells were incubated with anti-human C3d (1:50, Santa Cruz Biotechnology), then washed and incubated with Alexa-Fluor 555-conjugated secondary antibody for 1 h (5 μg/mL, Invitrogen).