Antibody-Dependent Cellular Cytotoxicity and Phagocytosis Assays

The ADCC and ADCP assays are also modifications of the CBA in combination with previously established flow cytometry-based ADCC (48 (link)) and ADCP assays (87 (link), 88 (link)). In this case, serum (1:10) or mAbs (1μg/mL unless otherwise indicated) were added to 5,000 MOG-GFP transfected HEK cells plated in 96-well round-bottom plates and incubated for 15 minutes at room temperature. All serum samples were HI prior to use to inactivate endogenous complement proteins. Then, effector cells were added at a 10:1 effector/target ratio. For the ADCC assay, the effector cells were NK cells that were magnetically isolated (EasySep Human NK Cell Isolation Kit, STEMCELL Technologies) from pooled HD-derived cryopreserved PBMCs. The effector cells for the ADCP assay consisted of the THP-1 macrophage cell line (ATCC), labeled with CellTrace Violet (Thermo Fisher Scientific). After addition of the effector cells, the plates were then incubated at 37°C for 4 hours and shaken intermittently. For the ADCC assay, the cells were then stained with Near IR Live/Dead (Invitrogen) stain for 30 minutes on ice, to identify killed target cells. MOG⁺ and MOG^– were detected as in the CBA as GFP⁺. For the ADCP assay, macrophages were identified in the V450 channel and phagocytosis of MOG⁺ cells in the GFP channel. Data were normalized to the mean media-only condition (no antibodies or serum).