Ros kit

Cells were incubated for 10 min in MACS buffer (PBS, 0.5% Fetal Calf Serum/FCS, 2 mM EDTA) containing 1 μg/ml of mouse Fc Block (Miltenyi Biotech) to prevent non-specific IgG binding to FcR-displaying leuKocytes. Cells were washed and stained for 30 min at 4°C. The following antibodies were purchased from BioLegend: GR-1 (APC or FITC), F4/80 (APC-Cy7), CD11a/CD18 (PerCP-Cy5) Dectin-1 (PE), CXCR2 (Alexa Fluor 647). The following antibodies were purchased from eBioscience: CD11b (PE-Cy7). Intracellular staining for MPO (PE) (Abcam) was performed on freshly sampled BAL cell suspensions. Cells were fixed and permeabilized using the Fixation & Permeabilization kit (eBiosciences). Reactive Oxygen species (ROS) from freshly isolated BAL neutrophils were quantified by flow cytometry using the ROS kit (Enzo). Data were acquired using a MACSQuant flow cytometer (Miltenyi Biotech). Samples were analyzed with MACSQuantify software (Miltenyi Biotech). All analysis was performed on total live cells in a sample.

Cells were grown on cover slides and treated as indicated in the manufacturer’s ROS kit instructions (Enzo-51011). This kit recognizes different reactive species (hydroxyl radicals, hydrogen peroxide, peroxynitrite) but does not detect superoxide. Transduced differentiated cells were washed twice and 1 ml of fresh differentiation media was added, with or without EGCG (10 mM) or H₂O₂ (100 μM) and incubated for 30 min at 37 °C. After additional wash and media renewal (1 ml), cells were incubated for 1 h at 37 °C with a solution composed 2X ROS detection and Oxidative stress reagent (5 mM; dilution 1:2500). Cells were then washed with 1X PBS and directly mounted using 15 μl of vectashield+DAPI, without any fixation. Pictures were taken using a DeltaVision Elite system (GE). An average of 100 stacks and 50 nuclei were taken per conditions. Images were then treated using IMARIS. Intensities of ROS foci and DAPI staining were used for analyses, excluding single-nuclei cells for myotubes analysis.