Anti ccr2 apc

Monocyte-derived macrophages or BMDMs were harvested and washed in cold PBS. Cells were incubated in Fc-block reagent (BD biosciences) and fixable viability dye eFluor 780 (Invitrogen) for 15 min at 4°C in cold PBS. Monocyte-derived macrophages were washed once and stained with the following antibodies: anti-CD62L PerCp-Cy5.5 (BD biosciences), anti-CCR2 APC (R and D Systems), anti-F4/80 efluor450 (eBioscience), anti-CSF1R BV711 (Biolegend), anti-Ly6G BUV395 (BD biosciences), anti-CD11b BUV737 (BD biosciences), anti-MHCII(I-A/I-E) PE (eBioscience) and anti-Ly6C PE-Cy7 (eBioscience). BMDMs were washed once and stained with the following antibodies: anti-CD45 FITC (eBioscience), anti-F4/80 BV421 (Biolegend), anti-CD11b BUV395 (BD biosciences). Stained cells were analyzed on by flow cytometry using a BD FACS CANTO instrument. Loss of eGFP or CD45 was assessed by gating on live F4/80 + macrophages using FlowJo X.

For flow cytometry analysis, isolated cells were pre-incubated for 10 minutes with purified anti-mouse CD16/32 (BioLegend) to block the Fc-mediated non-specific binding of antibodies. Then, cells were stained with the following antibodies: anti-CD64-PE/Dazzle 594, anti-CD115-PE/Dazzle 594, anti-CD3-PerCP/Cy5.5, anti-NK1.1-PerCP/Cy5.5, anti-Ly6G-PerCP/Cy5.5, anti-CD24-PE/Cy7, anti-CX3CR1-APC, anti-CCR5-APC, anti-Ly6C-APC/Cy7, anti-CD45-BV421, anti-I-A/I-E-BV605 (all from BioLegend), anti-CD19-PerCP/Cy5.5, anti-CD11b-AF700 (BD Biosciences, Franklin Lakes, NJ, USA), anti-CCR2-APC (R&D Systems, Minneapolis, MN, USA), and anti-F4/80-PE (Thermo Fisher Scientific) for 20 minutes on ice. After surface staining, dead cells were stained with Zombie aqua™ Fixable Viability Kit (BioLegend). For the intracellular staining of TLR7, cells were fixed and permeabilized with the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) according to the manufacturer’s instructions. Then, cells were stained with anti-TLR7-PE (BD Biosciences). Data were acquired on a FACS LSR Fortessa (BD Biosciences) and the percentage of each cell population and mean fluorescence intensity were analyzed using FlowJo software (TreeStar Inc, Ashland, OR, USA).