Fitc conjugated anti ly6g

Aortas were dissected, minced using scissors and enzymatically digested with 200 U/mL Liberase (Roche) and 40 U/mL DNase I (Sigma Aldrich) in HBSS plus 5% FCS for 1 h at 37°C. Cells were filtered through a 40 μm nylon strainer, washed with HBSS plus 5% FCS, collected by centrifugation at 400 g for 5 min at 4°C and then suspended in FACS buffer (PBS plus 0.2% FCS plus 1 mM EDTA). Murine Fc receptors were blocked using anti-CD16/32 antibodies (BioLegend) for 10 min on ice. Violet 510 Viability Dye (Cell Signaling Technology) was used to discriminate between live and dead cells. The cells were stained with the following antibodies for 30 min at 4°C: PerCP-Cy5.5-conjugated anti-CD45, APC-Cy7-conjugated anti-CD11b, FITC-conjugated anti-Ly6G, PE-Cy7-conjugated anti-F4/80, and APC-conjugated anti-Ly6C (all purchased from BioLegend). Flow cytometric analysis was performed using FACSCanto II flow cytometer (BD Biosciences) and DIVA Software (BD Biosciences).

Muscle tissues were digested first in DMEM solution containing 0.1% type II collagenase for 1 h at 37°C. The undigested tissues were then disrupted and filtered through a 70-μm nylon filter (Becton Dickinson, Franklin Lakes, NJ, USA) using a plunger. Isolated cells were resuspended in staining buffer (PBS with 0.5% bovine serum albumin) and incubated with different antibodies to detect leukocytes as described below. The antibodies utilized were as follows: PerCP-conjugated anti-CD45 for leukocytes, PE-conjugated anti-F4/80 for monocytes/macrophages, FITC-conjugated anti-Ly6G for neutrophils, and APC-conjugated anti-CD206 for macrophage subpopulations (all from BioLegend). Stained cells were analyzed with a FACS Canto II flow cytometer (BD Biosciences). Gated CD45-positive cells were considered to be leukocytes. Leukocyte subpopulations were presented as the percentages of macrophages (F4/80⁺) and neutrophils (Ly6G⁺) among leukocytes. The percentages of M1 (CD206⁻) and M2 macrophages (CD206⁺) among the macrophages were determined and the ratio of M1 to M2 macrophages was calculated.