(A) Pectoralis, abdominal and diaphragm muscles were dissected from adult Pax3GFP/+ (Ad), postnatal day 7 Pax3GFP/+ (P7) and adult mdx/mdx:Pax3GFP/+(mdx) mice and used for isolation by flow cytometry of Pax3(GFP)-positive satellite cells. After purification, adult, postnatal day 7 and mdx Pax3(GFP)-positive cells were used for RNA purification and transcriptome analysis (Pallafacchina et al., 2010). (B) RNA samples prepared from adult control (Ad), adult mdx (mdx) and P7 GFP-positive cells were analysed by qPCR for Myogenin transcripts relative to the level of Hprt transcripts, expressed as a log ratio. (C, D) Immediately after purification, adult control (Ad, quiescent, red), postnatal day 7 (P7, activated, blue) and adult mdx (mdx, activated, blue) GFP-positive cells were incubated with Cell Rox probe and analysed by flow cytometry to measure fluorescence intensity (f.i). (C) Representative results and (D) histogram quantification of Cell Rox fluorescence intensity (f.i.). (B, D) Error bars represent the mean ± s.d, with n = 3 independent animals (B) or n ≥ 6 independent animals (D), **p<0.01. (E–H) GFP-positive cells isolated by flow cytometry from the pectoralis, abdominal and diaphragm muscles of Pax3GFP/+ adult mice were cultured. (E–G) Mitochondrial respiration measured by oxygen consumption rate (OCR) in basal conditions and after addition of FCCP (maximal respiration) (E), and glycolysis measured by extracellular acidification rate (ECAR) in basal conditions and after addition of oligomycin (maximal glycolytic activity) (F) were simultaneously recorded on cells at D2-D4 of culture using the Seahorse XFe24 technology. (G) Histogram represents the OCR/ECAR ratio. (H) H2O2 production by freshly isolated (D0) and ex vivo activated satellite cells (D2–D3), was measured using amplex red technology. (E, F, H) Error bars represent the mean ±s.d, with n = 3 independent animals, *p<0.05, **p<0.01, ***p<0.001. (i) Tibialis Anterior (TA) muscles of wild-type adult mice were dissected 3 days after cardiotoxin injury. Cryo-sections were processed for immunofluorescence analysis with DAPI and antibodies directed against Pitx2 or Pitx3 (green) and MyoD (red). Scale bars, 20 μm. (J–L) GFP-positive cells isolated by flow cytometry from pectoralis, abdominal and diaphragm muscles of Pax3GFP/+ adult mice were cultured for the indicated days (D). (J) After 2, 3, 4 and 5 days of culture, cells were used for RNA extraction. Pitx2, Pitx3 and Myogenin transcripts were analysed by qPCR relative to the level of Hprt transcripts, expressed as a log ratio. (K, L) After 2, 4 and 5 days of culture, cells were used for immunofluorescence analysis with DAPI and antibodies directed against MyoD, Myogenin or Troponin T (TnT) (K), (L), red), and Pitx2 (K), green) or Pitx3 (L), green). White arrowheads indicate cells positive for Pitx and differentiation markers, arrowheads outlined in black indicate cells positive for Pitx2 or Pitx3 and negative for Myogenin, and stars indicate cells positive for Myogenin and negative for Pitx2. Scale bars, 20 μm. Representative images from at least 3 independent experiments are shown. (M) Pitx2flox/+:Pitx3+/-:Pax3GFP/+:R26RCre-ERT2/+ double heterozygote mice, obtained by 4-hydroxytamoxifen (4-OHT) intra-peritoneal (IP) injection on 5 consecutive days (D) and Pax3GFP/+ wild type mice were subjected to muscle injury by notexin injection into the pectoralis muscle. GFP-positive cells isolated from mice of both genotypes at D0, D2 and D3 after notexin injury were immediately incubated with Cell Rox probe and analysed by flow cytometry to measure fluorescence intensity (f.i). Histograms represent ROS quantification.