Ly51 6c3

We harvested the fractured callus of wild-type or Ccn2Acp5 mice on the 14th day after the fracture. Using a mortar and pestle, each sample was crushed separately and placed in separate 50-ml centrifuge tubes with 10 mL collagenase at 37°C for 30 min Each sample was subjected to continuous enzymatic digestion with collagenase and DNase at 37°C with gentle shaking for 30 min. The digestion step was repeated thrice. The digested cells were filtered through a 70-μm nylon mesh, centrifuged at 200 × g at 4°C, and resuspended in PBS containing 2% FBS. After the cells were washed with PBS, they were resuspended in 50 μL PBS containing 2% FBS. The pellet was stained using fluorescent dye-conjugated antibodies against CD45 (1:100, # 147718, Biolegend, United States), Tie2 (1:100, # 124009, Biolegend), CD51 (alpha V, 1:100, # 104105, Biolegend), Ly-51 (6C3, 1:100, # 108314, Biolegend), Ter119 (1:100, # 116228, Biolegend), CD105 (1:100, # 120406, Biolegend), CD200 (1:50, # 565547, BD Biosciences, United States), and Thy (CD90.2, 1:100, # 105335, Biolegend) for flow cytometry analysis.

Thymus tissue was enzymatically digested with Collagenase/Dispase (2.5 mg/ml; Roche) and DNase 1 (40 mg/ml; Roche). After digestion, CD45⁺ thymocytes were depleted using anti‐CD45 microbeads and LS columns (Miltenyi Biotec). Antibodies against the following were used to sort TEC populations: CD45 (30‐F11, eBioscience), EpCAM1 (G8.8, eBioscience), I‐A^b (AF6‐120.1, BD Bioscience), Ly51 (6C3, Biolegend), CD80 (16‐10A1, Biolegend). Reagents were conjugated to Pacific Blue, BV 421, BV605, PE, PerCP–eFluor 710, allophycocyanin–eFluor 780. Streptavidin PE‐Cy7 was used to detect biotinylated antibodies. Sorting was performed using a FACS Aria Fusion 1 sorter (BD) with a purity typically >98%.