Nonspecific igg

Infected cells were washed twice in PBS, and aliquoted into 96 well plates (2×10⁵ cells/well for PBMCs, 5×10⁴ for cell lines). Cells were then pelleted and resuspended in PBS containing 25 μg/ml of the appropriate anti-Env antibody, and incubated for 60 minutes at RT. The plate was then washed twice with PBS, and extracellular staining was performed with Anti-CD3-AF700 (Biolegend clone SK7), Anti-CD4-BV605 (Biolegend clone OKT4), anti-CD8 FITC (BD clone HIT8a), anti-Fc APC (Biolegend clone HP6017), and Live/Dead Blue (ThermoFisher) and incubated for 20 minutes at RT. Samples were then washed twice and cells were fixed with Fix/Perm solution (BD), washed twice with Perm/Wash (BD) and resuspended in Perm/Wash containing anti-gag KC-57-RD1 (Beckman) and incubated for 20 minutes. Cells were then washed twice with Perm/Wash and resuspended in PBS for data acquisition by flow cytometry. To avoid potential differences in env expression, all HC antibodies and controls were compared for recognition on the same batch of infected cells at the same time. Infected-cell recognition rates among each virus were Z-scored using the standardize function in Microsoft Excel. Negative controls include nonspecific IgG (Biolegend), IVIG, and PBS conditions. Positive controls include polyclonal HIVIG, and combinations of bNAbs directed against multiple binding sites (such as 10E8, PGT121, and 3BNC117).