Cd8a apc clone 53 6

At 24 h after osteotomy, blood and the fracture hematoma were analyzed for immune cells by flow cytometry. To remove erythrocytes from blood, lysis was performed twice for 5 min on 37°C using lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.125 mM ethylenediamintetraacetic acid (EDTA), all from Sigma-Aldrich). The hematoma between the bone ends was excised and pressed through a cell strainer to obtain a single-cell suspension. Cells of the innate and adaptive immune systems were stained using the following antibodies or respective isotypes: CD11b Alexa Fluor 700 (clone M1/70, eBioscience, 1:400 dilution), Ly-6G V450 (clone 1A8, BD Pharmingen, 1:400 dilution), F4/80 FITC (clone BM8, eBioscience, 1:50 dilution), CD3ϵ PE-Cyanine 7 (clone 145-2C11, eBioscience, 1:100 dilution), CD4 APC-eFluor 780 (clone GK1.5, eBioscience, 1:200 dilution), CD8a APC (clone 53-6.7, eBioscience, 1:800 dilution), and CD19 PE (clone 1D3, eBioscience, 1:400 dilution). 7-AAD (Sigma) was used to distinguish between living and dead cells. A minimum of 10,000 events was measured on an LSRII flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (FlowJo, Ashland, OR, USA). Detailed gating strategy is provided in Supplementary Figure 1C.

C57BL/6 mice were immunized twice by a subcutaneous injection of rOVA (30 μg), rlipo-OVA (30 μg) or PBS at one-week intervals. Seven days after the final immunization, splenocyte CD8⁺ T cells producing IFN-γ were determined by intracellular cytokine flow cytometry. The peptides used to stimulate the cells were added at a concentration of 2 μg/ml for 18 h at 37°C; then, 1 μl/ml of Brefeldin A (eBioscience), 1 μg/ml ionomycin (Sigma-Aldrich) and 10 μg/ml phorbol 12-myristate 12-acetate (PMA) were added for an additional 4 h before harvesting the cells from the culture. The cells were subjected to intracellular cytokine (IFN-γ) staining using the Intracellular Fixation & Permeabilization Buffer Set (eBioscience) according to the manufacturer's instructions. The cells were washed once with FACS buffer (PBS, 2% FBS and 0.05% sodium azide) and stained with the following monoclonal antibodies: CD16/CD32 (clone 93, eBioscience), CD3ε-PE (clone 145-2c11, Biolegend), CD8a-APC (clone 53–6.7, eBioscience), IFN-γ-PE-Cy7 (clone XMG1.2, eBioscience) and the isotype control antibody (rat IgG1k, eBioscience). Sample acquisition was analyzed using the FACSCalibur (BD Biosciences).