Fitc anti mouse c kit

Isolated cells were incubated with 5% rat serum for 15 min at 4 °C. The cells were then incubated with primary antibody cocktails for 30 min at 4°C in the dark (FITC anti-mouse c-Kit, Biolegend, Cat# 105806, 1 μl in 50μl cocktail volume; PE anti-mouse CD34, Biolegend, Cat# 119308, 2 μl in 50μl cocktail volume; BV421™ anti-mouse lineage cocktail, Biolegend, Cat# 133311, 10 μl in 50μl cocktail volume; PE/Cy7 anti-mouse Sca-1, Biolegend, Cat# 108114, 1 μl in 50μl cocktail volume; PerCP-eFluor 710 anti-mouse CD135, eBioscience, Cat# 46–1351-82, 1 μl in 50μl cocktail volume; APC anti-mouse CD16/CD32, eBioscience, Cat# 14–0161-82, 1 μl in 50μl cocktail volume; PE-CF594 anti-mouse CD127, BD Biosciences, Cat# 562419, 1 μl in 50μl cocktail volume). After washing, the cells were then stained with flexible viability dye eFluor 780 (1 ul/ml, eBioscience, San Diego, California, USA) for 30 min at 4°C, washed with PBS twice, and fixed with 1% PFA for flow cytometry.

Adherent BMCs were digested with Accutase (Millipore), suspended in growth medium, pelleted by centrifugation at 1200 rpm for 5 min. Cells were resuspended in 0.1% BSA-PBS at a concentration 1×10⁸ cells/ml for magnetic bead separation using EasySep kits (Stem Cell Technology) by labeling cells with APC or FITC -anti-mouse c-Kit, PE -anti-mouse CD11b, or APC -anti-human CD14 (Biolegend). For FACS analysis, 1×10⁶ cells were labeled with APC-anti-mouse CD11b, APC/Cy7-anti-mouse F4/80, PE-anti-mouse-RANK (CD256), Brillant^Violet421-anti-mouse CD115, PE-anti-human CD33, APC-anti-human CD34, FITC-anti-human CD14, Brillant^Violet421-anti-human CD16, and analyzed by MACQuant Analyzer (Miltenyi Biotec).