Apc conjugated anti cd32 fun 2

Peripheral blood mononuclear cells (PBMCs) were isolated from freshly heparinized blood by centrifugation on a Ficoll-Hypaque gradient (Pharmacia, Uppsala, Sweden). 50-100 million HIV-1 infected patient PBMCs were first incubated with Fc receptor blocking solution (TruStain FcX; Biolegend, San Diego, California, USA) for 10 min and then labeled with the following antibodies: BV510-conjugated anti-CD3 (OKT3; Biolegend), PerCPconjugated anti-CD4 (SK3; Biolegend), PE-Cy7-conjugated anti-CD19 (SJ25C1; BD Pharmingen, San Jose, California, USA), APC-conjugated anti-CD32 (FUN-2; Biolegend), FITC-conjugated anti-CCR7 (150503; R&D Systems, Minneapolis, Minnesota, USA), PE-conjugated anti-CD14 (HCD14; Biolegend), BV421-conjugated anti-CXCR5 (RF8B2; BD Pharmingen) and BV510-conjugated anti-CD45RA (HI100; Biolegend). Total CD4 + cells and CD4 + CD19 + cells (excluding CD14 + cells) were sorted using an ARIA II cell sorter (BD Biosciences, Franklin Lakes, New Jersey, USA). The purified CD4 + CD19 + cells were dissociated using 2 mmol/l eathylene diamine tetraacetic acid and vigorously vortexed according to the previously described protocol [9] . The single cell suspension was analyzed for phenotypic expression using FACSVerse (BD Biosciences) and the data were further analyzed using the FlowJo software (TreeStar, Woodburn, Oregon, USA).