Fig. 4.

ADCC assay using cells spinoculated with AT-2 inactivated HIV-1 BaL virions. Left panel. EGFP-CEM-NKr-CCR5-SNAP cells were labeled with Alexa Fluor 647-SNAP tag and then spinoculated with HIV-1 Bal AT-2 virus at 2000 rpm for 2 h at 12 °C. After two washes, cells were incubated with dilutions of mAbs (C11, N5-i5, N12-i2 or Synagis) for 15 min at RT, then PBMC were added to the reaction for 3 h at 37 °C. At the end of the incubation, the samples were washed with PBS, fixed with 1% paraformaldehyde and analyzed by flow cytometry. The ADCC results are representative of three independent assays and the bars indicate the range of the values of cytotoxicity of duplicate samples. Right panel. The efficiency of the spinoculation was evaluated by cell surface staining with 2 μg/ml Alexa Fluor-594-conjugated mAbs C11 (Panel A), N5-i5 (Panel B) or N12-i2 (panel C).