Cd14 pacblue

Single-cell suspensions of splenocytes were stained with LIVE/DEAD Fixable Violet Dead Cell Stain (Life Technologies) according to the manufacturer’s instructions. Cells were washed with PBS and stained with CD3-APC-H7, CD4-APC, CD8-PERCP-Cy5.5, CD16-Alexa Fluor 700, CD14-PacBlue and HLA-ABC-PECy7 (BD Biosciences) for extracellular stains. For intracellular detection of HIV-1 core Ag p24 cells were fixed and permeabilized using BD Cytofix/Cytoperm Solution Kit (BD Biosciences) according to the manufacturer’s instructions before staining with 1 μl per sample of HIV-1 core Ag–RD-1 (Beckman Coulter). When cell number was limiting, a minimum of 5 × 10³ live gated events were collected in each sample and analyzed to maintain cell number between samples for comparison of different T cell populations. Data were collected on an LSR II flow cytometer with BD FACSDiva software (BD Biosciences) and analyzed using FlowJo Software (TreeStar, Ashland, OR).

CD14⁺ cells were purified from PBMC using auto-MACS Pro by positive-selection. Monocyte purity was measured by flow cytometry after staining with mouse-antihuman monoclonal antibodies against CD14-PerCP (BD Biosciences) and CD3-PacBlue (BD Biosciences). Isolated CD14⁺ monocytes (2 × 10⁵ cells/200 µl) were cultured in RPMI 1640 medium (Life Technologies), supplemented with 10% FBS and 1% P/S. Monocytes were cultured with MP, or MPγ at different ratios (1:10,000, 1:40,000, 1:80,000) in polypropylene tubes. After 24 h of incubation, monocytes were collected for PCR analysis or flow cytometry after staining with CD14-PacBlue, CD3-PerCP, CD16-FITC, PD-L1-PE and CD90-APC (all BD Biosciences).