Anti mouse cd206 c068c2

For surface markers analysis, live cells were re-suspended in 1 × PBS and stained with anti-mouse CD45 (30-F11, Biolegend), CD11b (M1/70, R&D Systems), F4/80 (BM8, Biolegend), CD86 (GL-1, Biolegend), MHC-II (M5/114.15.2, Biolegend), CD117 (2B8, Biolegend), CD115 (AFS98, Biolegend), CD16/32 (93, Biolegend), CD34 (HM34, Biolegend), Sca1 (D7, Biolegend), SIRPα (P84, biolegend), Lin (Stem Cell), and APC-Cy7 Streptavidin (Biolegend) at 4°C for 30 min. The concentration at each antibody was used as the product protocol recommended. For intracellular cytokine staining, cells were fixed and permeabilized with Fixation and Permeabilization Kit (eBioscience) at room temperature for 40 min and labeled with anti-mouse CD206 (C068C2, Biolegend). Multicolor FACS analysis was performed on an LSR Fortessa Analyzer (BD Biosciences). All data analysis was performed using the flow cytometry analysis program FlowJo-V10.

M279 (Amgen) is a rat IgG mAb, which blocks ligand binding to the CSF-1R. In this study, mice were treated with 200 µg M279 or rat IgG (Sigma-Aldrich) intraperitoneally three times a week for 9 wk. Manocept–Alexa Fluor 488 (Navidea Biopharmaceuticals) is a fluorescently labeled derivative of FDA-approved 99MTC-Tilmanocept targeting MR. Naive mice were injected with Manocept–Alexa Fluor 488 intradermally and intravenously and sacrificed 1 and 2 h later, respectively, for analysis. Naive mice were also injected intravenously with 1% (wt/vol) of 2-MD FITC-dextran (Sigma-Aldrich) or 10 µg of anti–mouse CD206 (C068C2; Biolegend) and sacrificed 10–30 and 3 min later, respectively. For measuring apoptotic cell uptake, 10⁶ thymocytes were treated for 6 h with 1 µM dexamethasone (Sigma-Aldrich) and were intradermally injected into naive mice.