Anti cd29

Flow cytometry was performed to determine the phenotype of MSCs. MSCs were washed twice with washing buffer (PBS containing 3% FBS) and incubated for 30 min on ice with the following fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)-conjugated antibodies in the washing buffer: anti-CD29 (1:200,eBioscience, San Diego, CA, USA), anti-CD44 (1:300,eBioscience), anti-CD45 (1:400,eBioscience), anti-CD90 (1:400,eBioscience), anti-CD34 (1:50, Abcam, Cambridge, MA, USA), and anti-CXCR4 (1:50, Abcam) antibodies. The MSCs were rinsed with the washing buffer again, and a FACSCalibur system (BD Biosciences, San Diego, CA, USA) was used for characterizing MSCs. Data were analyzed using the FlowJo software (Treestar, Ashland, OR, USA).

Flow cytometry was used to identify the phenotypes of the cultured MSCs. MSCs at passage 3 were incubated with anti‐CD29, anti‐CD44, anti‐CD45, anti‐CD90 (eBioscience, San Diego, CA, USA) or anti‐CD34 (Santa CruzBiotechnology, Santa Cruz, CA, USA). Antibodies of the corresponding isotype served as the negative control. To detect CXCR4 expression on the cellular surface, cells were stained with mouse anti‐rat CXCR4 antibody (Sigma, St. Louis, MO, USA) or the isotype control. The secondary antibody was donkey anti‐mouse IgG PE (Proteintech, Chicago, IL, USA).

Immunostaining was performed as previously described^{9 (link), 24 (link)}. Briefly, Mac-ASCs and Sat-ASCs (P1) were harvested by centrifugation, washed once with PBS, and re-suspended in PBS. Approximately 10⁵ cells in 1 mL of PBS were incubated with 5 μL of anti-CD106, anti-CD105, anti-CD29, or anti-CD90 antibodies (eBioscience, CA, USA) for 20 min. Then, the cells were washed and re-suspended in 1 mL PBS, and the expression of cell surface markers was analysed by flow cytometry.

Surface antigens were detected by direct immunofluorescence using flow cytometry (Beckman Coulter, Mississauga, Ontario, Canada and LSRFortessa Cell Analyzer (BD Biosciences, San Jose, California, USA). Briefly, control cells and cells exposed to arsenic were washed twice with PBS supplemented with 5% FBS (Wisent) and 0.01 M sodium azide (flow cytometry buffer). The cells were then exposed to labeled anti-CD29 (eBioscience anti-murine; Pharmingen anti-human: HUTS-21 clone), P-selectin (BD Pharmingen), CD14 (eBioscience), CD41 (BD Pharmingen), Ly6G (eBioscience) or their specific isotype control antibody and incubated for 45 min on ice in the dark. Cells were then washed twice with flow cytometry buffer, fixed in 2% paraformaldehyde and analyzed by flow cytometry. The gates for positive-staining cells were determined by comparison with cells stained with the isotype-matched control antibodies. The FCS express and FlowJo softwares (denovosoftware, CA, USA; flowJo LLC, Ashland, Oregon, USA) were used to analyze the data.

Differentiated myoblasts were phenotyped and sorted by flow cytometry (Sue and Bill Gross Stem Cell Institute, University of California-Irvine, Irvine, CA). Briefly, myogenic precursors at days 35 and 49 were dissociated with dissociation buffer (ThermoFisher Scientific) and stained with cell surface pluripotency marker, anti-CD34, and mesenchymal stem cell (MSC) markers anti-CD73, anti-CD105 and anti-CD29 (Life Technologies) and skeletal muscle marker, anti-CD56 (ThermoFisher Scientific) as well as isotype controls (BD Pharmingen). Dead cells were excluded by propidium iodide (Sigma-Aldrich) and samples were analyzed using FACSAria II and FACS Diva software (BD Biosciences, Franklin Lakes, NJ).