Ly6g bv605

1 × 10⁶ cells were Fc blocked (Biolegend101320) and stained for 1 hour, on ice, in the dark, as previously described.^{20 (link)} The following antibodies were used, B220−FITC (BD553087), I Ab PerCP−Cy5.5 (Biolegend116416), Ly6C PE or PECy7 (Biolegend128008, 128071), HLA−DR PECy7 (eBioscience25−9956−41/Biolegend307606), CD11b BV510 (BD562950/Biolegend101263), Ly6G BV605 (BD563005/Biolegend127639), CD11c APC (BD550261), CD3 A700 (eBioscience56−0032−82/Biolegend100216), Live Blue Fluorescent reactive dye (LifeTech L34962), and CD45 BV650 (Biolegend103151).

Lung tissue was chopped and digested with collagenase-D (1 mg/ml; Roche) and DNase I (10 mg/ml; Sigma-Aldrich) for 1 h at 37°C with agitation. To detect cytokines, brefeldin A (5 µg/ml) was added during the digest. Lungs or spleens were passed through a 70-µm cell strainer to a obtain single-cell suspension, followed by RBC lysis with ACK buffer. The cells were incubated with Fcγblock (anti-CD16/CD32 antibody, BD Biosciences) (1:100) to block IgG Fc receptors. Cells were incubated with LIVE/DEAD Aqua (Invitrogen), followed by surface staining with fluorochrome-conjugated anti-mouse Abs for various markers. The following surface Abs were used: CD69-FITC, CD3-APC-ef780, MHCII-APC, Ly6C-PerCP-Cy5.5, CD86-FITC, CD11b-APC-ef780, CD38-ef450, F4/80-PE-Cy5, CD49d-PerCP-ef710, CD3-AF700, CD8-APC-ef780, CD44-PE-Cy7, CD4-PE-Cy5 (eBiosciences), CD44-BV605, CD4-BV785, CD103-PE, Ly6G-BV605, CD80-PE-Dazzle594 (BioLegend), CD62L-PE-CF594, SiglecF-PE, CD103-BV786 (BD Biosciences). For detection of intracellular cytokines, cells were fixed in 2% PFA and permeabilized with 0.5% saponin (Sigma-Aldrich, Ireland), followed by staining with IL-17A–V450 (BD Biosciences). Fluorescence minus one samples were used as controls. Flow cytometric analysis was performed on an LSR Fortessa, and data were acquired using Diva software (BD Biosciences). The results were analyzed using FlowJo software (TreeStar).

Cell infiltration into muscles was analyzed by flow cytometry. 1 week after CCI, C57BL/6 J mice were sacrificed via cervical dislocation; the right biceps femoris and gastrocnemius muscles were collected, minced, and treated with collagenase. Cells were isolated, washed twice with phosphate-buffered saline, incubated for 8.5 min in erythrocyte lysis buffer, and finally suspended in phosphate-buffered saline. Next, isolated cells were incubated with an Fc Block antibody (BD Biosciences, Bedford, MA, USA) for 15 min on ice, labeled with dye-conjugated antibodies (CD45-FITC, 25 μg/mL; Ly6G-BV605, 10 μg/mL; F4/80-PE, 10 μg/mL; CD11b-BV711, 2.5 μg/mL; CD301-Alx647, 2.5 μg/mL; Ly6C-BV421, 2.5 μg/mL; BioLegend), and analyzed by flow cytometry using BD LSR Fortessa (BD Biosciences) and FlowJo V10 (Tomy Digital Biology, Tokyo, Japan). DRAQ7 (BioLegend) was used to exclude dead cells.

After the mice were sacrificed, the lungs were removed, washed with PBS, cut into small pieces using a scalpel, transferred into GentleMACS C-tubes (Miltenyi Biotec, Germany), and processed using the lung dissociation kit and a GentleMACS dissociator (Miltenyi), according to the manufacturer’s instructions. Homogenized lungs were passed through a 70-mm nylon mesh to obtain a single-cell suspension. The resultant cells were counted using an automated cell counter (EVE, NanoEnTek, Seoul, South Korea) and processed for flow cytometry, as detailed previously^{94 (link),95 (link)}. Briefly, cells were pre-incubated with anti-mouse CD16/32 mAb (Biolegend) to block FcγIII/II receptor sites and then stained with Zombie Aqua Viability dye (Biolegend). After washing, the cells were stained with a mixture of fluorochrome-conjugated antibodies using the following panel of mAbs: CD45-Alexa Fluor 700, CD11b-Alexa Fluor 488, Ly6G-BV605, Ly6C-APC-Cy7, F4/80-PE, and MHC II-BV785 (all from Biolegend). Data were collected on 30,000 cells per sample using FACS Celesta (BD) and analyzed using BD FACS Diva software (BD Bioscience).