Cd3 a700

Intracellular staining was used to assess the effector functions of activated natural killer (NK) cells isolated by negative selection (RosetteSep kit, Stemcell technologies) from fresh whole blood from healthy donors, as previously described (24 (link)), with the following modifications – 50 μl of 0.25 mg/ml IgG purified from plasma (data not shown) or 50 μl CVL were incubated with the one of four HIV antigens Con6 gp120/B (Consensus Group M gp120), gp41 (Ectodomain) (HIV-1), p66 RT, and p24 (HIV-1/Clade B/C CN54) (Immune Technology, California, USA or ImmunoDX, Massachusetts, USA) and 50,000 NK cells/well. Following incubation with monensin and Brefeldin A as well as CD107a, cells were stained with CD3⁻ A700, CD56 PE-Cy-7, CD16 APC-Cy7, IFN-γ APC, and MIP1-β PE (BD Biosciences, California, USA) and were then fixed with BD cell fix and then acquired on a LSR Fortessa (BD Biosciences, California, USA). Enriched NK cells were acquired and selected for singlets, followed by CD3 negative lymphocytes that were CD56⁺/CD16⁺ (Figure 2). The release of CD107a, IFN-γ and MIP-1β was used to assess frequency of activated NK cells to determine NK-cell activated ADCC (Figure 2) (9 (link)). When calculating HIV-specific antiviral activities, the non-specific ADCC activity obtained as background activity from PBS/NHS was subtracted from HIV-specific antibody-mediated ADCC activities.