Cd16 bv785

ELISA plates were coated with 50mL of 2μg/mL PPD overnight at 4C. Coated plates were blocked with 5% BSA for 1hr at RT and washed 3x with PBS before 50μL of diluted serum (1:30, 1:100, 1:300, 1:1000) was added to each well. Serum dilutions were incubated 2hrs at 37C on antigen-coated plated and washed prior to adding 5x10⁴ NK cells per well, isolated from healthy HIV^- donor buffy coats by RosetteSep (Stem Cell 15065). CD107a-BV605 (BioLegend 328634), 5μg/ml brefeldin A (BioLegend 420601), and 0.7μl/mL GolgiStop (BD 554724) were also added to each well and incubated for 5hrs at 37C. Following this incubation, NK cells were surface-stained with CD16-BV785 (BioLegend 302046), CD56-PE-Cy7 (BD 335791), and CD3-APC-Cy7(BioLegend 300426). An intracellular stain was then performed using Perm/Fix Solution (BD 554714) with IFNγ-PE (BioLegend 506507) and anti-MIP1β-BV421(BD 562900). Samples were fixed with 4% and NK cell activation was analyzed on the iQue Screener. AUC frequencies of NK cells bearing CD107a, expressing IFNγ and MIP1β across, were derived from the signal across the dilution series tested in the donor cells.

PBMCs in suspension were incubated in PBS with 0.1% (v/v) heat-inactivated FBS with optimally titrated concentrations of the antibodies CD3-AF700 (BioLegend, San Diego, CA, USA—UCHT1), CD14 -APC-Cy7 (BioLegend—HCD14), CD16-BV785 (BioLegend−3G8), CD19-APC-Cy7 (BioLegend—HIB19), CD56-BUV395 (BD Biosciences, San Jose, CA, USA—NCAM16.2), CD57-PEDazzle594 (BioLegend—HNK-1), KIR2DL1/KIR2DS5-PE (R&D Systems, MN, USA−143211), KIR2DL2/S2/L3-BV650 (BD Biosciences—DX27), KIR3DL1-BV421 (BioLegend—DX9), NKG2A-PECy7 (Beckman Coulter, CA, USA—Z199), and LIVE/DEAD fixable near-IR dye (Thermo Fisher Scientific) for 20 min at 4°C in the dark. Samples were subsequently washed in PBS then fixed with 0.5% (w/w) PFA (Sigma-Aldrich, MO, USA) before acquisition on a BD LSRFortessa flow cytometer.