Cd11b biotin

Cells to be stained were resuspended in FACS buffer (HBSS containing 1% FCS) and incubated with the indicated antibodies for 15 minutes on ice. Cells were then washed in FACS buffer before acquisition on an LSR-II flow cytometer (BD Bioscience, Franklin Lakes, NJ) and analysis using Flowjo (Treestar). Antibodies (Biolegend, San Diego, CA unless otherwise stated) used were anti-mouse CD4 PerCP-Cy5.5, CD8 Pacific Blue/APC-cy7, PD-1 FITC, CXCR5-biotin (BD Bioscience), CD44 Pacific Blue, GL-7 FITC, FAS PE, CD138 APC, CD19 APC-cy7, CD23 PE, CD21 PerCP-Cy5.5, CD11b-biotin, CD11c Pacific Blue/APC, B220 PE, PDCA-1 Pacific Blue and streptavidin APC/FITC/PerCP. For intracellular staining of markers, an intracellular staining kit (Fix/Perm, eBioscience, San Diego CA) was used together with anti-mouse Foxp3 PE (eBioscience).

To isolate enough number of NG2⁺ bone mesenchymal stromal cells for transplantation, the whole bone cells were prepared and pooled from the bones (femur, tibia, calvarium, sternum bones) of nine NG2-CreERTM^+/−;tdRED^+/+ female mice. To facilitate cell sorting, the majority of immune cells were pre-depleted by MACS sorting. Briefly, whole bone cells were incubated with CD45-biotin, CD11b-biotin, CD3e-biotin, Ly-6C/G-biotin and TER-119-biotin (BD Bioscience, Cat# 559971) in PBS with 2% serum, 1% antibiotics for 15 minutes at 4°C. After washing, cells were re-suspended and incubated with Streptavidin-bound magnetic beads (BD Bioscience, Cat# 557812) for 15 minutes at 4°C. Then cells were rinse twice and the biotin negative cells were collected by EasySep^™ Magnet (StemCell, Cat# 18000). The CD45⁻CD31⁻TER119⁻DAPI⁻NG2-tdRED⁺ cells were immediately sorted from the enriched population by a BD Aria II cell sorter with 100 μm nozzle. About 120000 cells in total were collected and then re-suspended in 60μl PBS. Purified cells were directly transplanted into the femur bones of five 8-week-old female C57BL/6 mice (10μl per animal) through intra-femoral injection with a 28G BD insulin syringe. As sham controls, 5 age-matched female C57BL/6 mice were injected with 10μl PBS.