Pe cy7 anti mouse cd11b clone m1 70

The purified neutrophil suspensions were centrifuged, resuspended in FACS buffer (1x PBS with 1% BSA), and blocked with anti-mouse CD16/CD32 antibody (Fcγ III/II, BD Biosciences) for 10 min on ice. After blocking, cells were incubated with the following monoclonal antibodies for 30 min at 4°C in the dark: PE-Cy7 anti-mouse CD11b (clone M1/70, BioLegend), FITC anti-mouse Ly6G (clone 1A8, Biolegend), Percp-Cy5.5 anti-mouse CXCR2 (clone TG11, Biolegend), APC anti-mouse CXCR4 (clone 2B11, BD Bioscience). Data were acquired on a FACS Canto II (BD Biosciences), and analysis was performed with FlowJo software (Ashland, USA). Neutrophils were identified as CD11b⁺Ly6G⁺, CXCR2⁺/CXCR4⁻ cells were fresh neutrophils, and CXCR2⁻/CXCR4⁺ cells were aged neutrophils.

Pools of 20 limb muscles of SCID CB17 mice (Tibialis anterior and Gastrocnemius) were harvested 7 days after myoblast injection and processed as a single sample (n = 4–7 samples/group). Tissues were minced, digested with DNase (0.2 mg/ml, Sigma-Aldrich), Collagenase type IV (484 U/ml, Worthington), Collagenase I (0.5 mg/ml; Sigma-Aldrich), and Collagenase II (0.5 mg/ml, Sigma-Aldrich) in low-glucose DMEM (Sigma-Aldrich) for 45 min at 37°C under constant shaking, filtered through 100- and 70-μm cell strainers (BD Biosciences), and washed twice in PBS. Cells were stained at 4°C for 30 min with the following fluorescently labeled antibodies: PE-anti-mouse CD31 (clone 390; BioLegend, San Diego, CA, USA) at 1:200; PE-Cy7-anti-mouse CD11b (clone M1/70, BioLegend) at 1:1,000. CD31⁺ and CD11b⁺ cells were isolated with an Influx cell sorter (BD Biosciences). RNA was extracted, and gene expression analysis was performed as described below.