Anti ccr5

Murine BM cells were cultured in the presence of M-CSF and different doses of LPS as described above. In some experiments, control or IRF5 siRNA (30 pmol, Life Technologies) was also added to cell cultures. After 5 days, cells were harvested and stained with anti-CD11b, anti-Ly6C, anti-Ly6G, anti-CCR5, anti-TLR4, and anti-CD14 antibodies (BioLegend). Propidium iodide (PI) was also added to determine the cell viability. To detect the production of IL-12, BM cells cultured for 5 days were treated with PMA (20 ng/ml), ionomycin (1 μg/ml), and GolgiStopTM protein transport inhibitor (BD Biosciences) for 4 h, and then stained with anti-Ly6C, anti-Ly6G, and anti-CD11b antibodies. After fixation and permeabilization using Cytofix/CytopermTM kit (BD Biosciences), cells were stained with anti-IL-12 antibody (BD Biosciences). The cell phenotype was then analyzed by flow cytometer. The data were processed by FACSDiva or Flow Jo.

The expression of chemokine receptors was determined on the surface of fresh monocytes or cultured monocytes^{53 (link), 54 (link)}. Cells were incubated for 20 min at room temperature in the dark with anti-CD14, anti-CD163 (BDBioscience) anti-CD16 (Immunotools, Friesoythe, Germany), anti-CCR2 (R&D Systems, Weisbaden, Germany), anti-CCR5 (Clone:HEK/1/85a), anti-CXCR4, anti-CX3CR1, anti-CD206, and anti-HLA-DR (Biolegend, San Diego, CA) monoclonal-antibodies. Red blood cells were then lysed using RBC lysis buffer (1X) (Biolegend). Cells were washed twice with staining buffer (PBS, supplemented with 0.5% BSA; Calbiochem Merck, Darmstadt, Germany) and resuspended in 200 μl of staining buffer to be analyzed by flow cytometry.
The surface expression of different markers was analyzed on gated CD14+ monocytes and on gated monocyte subsets (identified as CD14+ CD16- (classical), CD14+ CD16+ (intermediate) and CD14lowCD16++ (non-classical)) using a MACSQuant® Instrument (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The percentage of positive cells (% cells) and mean fluorescence intensity (MFI) of each individual marker were calculated using © FlowJo, LLC 2013-2016 data analysis software.