Anti cd66b pe

Cells were washed twice in phosphate-buffered saline with 1% bovine serum albumin and 1:200 of an unlabeled blocking anti-CD32a antibody, and an excess of unlabeled mouse IgG, and then stained with a mixture of antibodies against surface antigens: anti-CD66b (PE/Cy7-labeled, Biolegend #305115, San Diego, CA, USA) at 1:200, anti-CD16 (PerCP-labeled, clone 3G8 Biolegend #302029) at 1:200, anti-CD14 (PE-labeled anti-human CD14 antibody clone 63D3 (Biolegend #367103) at 1:200, anti-CD19 (APC/Cyanine7-conjugated, clone HIB19, Biolegend #302218) at 1:200, and anti-CD15 (PerCP-conjugated, clone W63D, Biolegend #323018) at 1:200 in phosphate-buffered saline with 1% bovine serum albumin for 30 min at 4 °C in the dark. Affinity-purified patient anti-Env and anti-cit-Env antibodies, or the human mAb L204:01A01, were conjugated to varying AlexaFluor labels according to AlexaFluor Microscale Protein Labeling Kits (Invitrogen A30006, A30009) and added to the cells for 30 min at 4 °C in the dark, washed twice with phosphate-buffered saline, 1% bovine serum albumin, and resuspended in 200 µL of this buffer for analysis on a CytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA, USA). These data were analyzed using BD Bioscience’s FlowJo v10.1 software package.