Anti cd14 apc h7 clone mφp9

Each sample was aliquoted (300 µl) into one tube and stained with anti-IgE-PE (clone BE5, EXBIO Praha, Vestec, Czech Republic), anti-IgG-FITC (clone G18-145, BD), anti-HLA-DR- PerCP-Cy5.5 (clone G46-6, BD), anti-CD123-APC (clone AC145, Miltenyi Biotec; Bergisch, Gladbach, Germany), anti-CD16-PB (clone 3G8, Biolegend), anti-CD14-APC-H7 (clone MφP9, BD) and anti-CD45-krome orange (clone J.33, Beckman Coulter) for 15 min in the dark at RT. Then samples were incubated with 2 ml of FACS Lysing solution (BD) for 10 min in the dark at RT and centrifuged for 5 min at 540 g. The supernatant was discarded and the cell pellet was washed twice in 2 ml of PBS with centrifugation of 5 min at 540 g, resuspended in 0.5 ml of PBS, and stored at 4 °C before acquisition.

Trogocytosis was measured using a previously described assay (45 (link)). CEM.NKR.CCR5 cells were washed with PBS and stained with PKH26 (Sigma-Aldrich, St-Louis, MO, USA) at 2μM in Diluent C at RT for 5 min. Cells were then washed with R-10, resuspended in R-10, and incubated with WT gp120 or ΔV1 gp120 for 1 h at RT in 96-well polypropylene plates. Cells were washed twice with R-10 and incubated with 300-fold diluted plasma samples. Cryopreserved healthy control PBMC were next added in R-10 at an effector to target (E:T) cell ratio of 50:1 and then incubated for 5 h at 37°C. After the incubation, cells were washed, stained with live/dead aqua fixable stain and anti-CD14 APC-H7 (clone MΦP9, BD, San Jose, CA, USA), washed again, and fixed with 4% formaldehyde (Tousimis, Rockville, MD). Fluorescence was evaluated on an LSRII flow cytometer (BD Biosciences). Trogocytosis was evaluated by measuring the PKH26 mean fluorescence intensity of the live CD14⁺ cells.