Figure 2.

Schematic diagram of the antibody-dependent cell-mediated cytotoxicity (ADCC) assay. ADCC activity was determined by a flow cytometry-based assay using two fluorescent dyes. PKH-67, a membrane-labeling dye, was used to specifically identify the HA-transfected target cells. 7AAD was excluded by viable cells but could penetrate the cell membrane of dead or dying cells and intercalate into double-stranded DNA. Briefly, 50 μl of PKH-67-labeled target cells (10⁶ cells/ml) was dispensed into a round-bottom 96-well plate, followed by addition of E1/E2/HA/PBS sera and effector/PBMC cells. Following a 3-h incubation, 7-AAD was added. Cell death was determined on a FACSAria III flow cytometer using BD FACS Diva software (BD Biosciences). Percent cell death was analyzed by the Flowjo software.