Cd16 af700

Frozen PBMCs were thawed and distributed into three tubes for antibody staining. All tubes contained antibodies to the following markers: CD14-FITC (Southern Biotechnology Associates), CD16-AF700 (BD Bioscience), and CD3- and CD19-PerCP.Cy5.5 (eBioscience). Additional antibodies included: Tube 1, CD11b-APC-Cy7 (eBioscience) and CD32-PE (BD Bioscience); Tube 2, CD64-PE (BD Bioscience); and Tube 3, CD32B-PE detected with goat anti-rabbit PE (AbCAM). Proper isotype controls were included for each primary antibody. All incubations were done in ice. Fc-receptors were blocked with human IgG (Sigma-Aldrich) for 20 min, followed by addition of antibodies for 30 min in PBS with 1% bovine serum albumin, 0.1 mM EDTA and 0.02% sodium azide. Prior to analysis 7AAD (BD Bioscience) was added to each tube. Acquisition was conducted in a FACS CANTO-II using FACS DIVA v6.0 (BD Biosciences). Monocytes were identified based on their forward and side scatter properties and then dead (7AAD-positive), CD3 and CD19-positive cells were excluded. Further details on the gating strategy are provided in the Supplement (Fig. S2). After identifying monocyte sub-populations based on CD14 and CD16 expression, the median fluorescence intensities of each fluorochrome was established.

The following antibodies were used in flow cytometry: CD45-V500 (clone HI30), CD3-BV786 (clone OKT3), CD4-Vioblue (clone RPA-T4), CD8-APC780 (clone SK1), CD19-APC, CD15-PerCP-Cy5.5, CD14-ECD, CD16-AF700, CD11b-PE-Cy7, CD209-FITC, CCR5-PC5, CD163-BV605, and major histocompatibility complex, class II, DR (HLA-DR)-PE (all BD). The cells were acquired using an LSRFortessa flow cytometer and FACSDiva software (BD). Data were analyzed using FlowJo software. At least 10,000 cells were acquired for each analysis, and each representative flow plot was repeated more than three times.