Anti sca 1

The cell suspension was preincubated with Fc block (BD Biosciences) to avoid nonspecific binding of antibodies. The following primary antibodies were used: anti-CD3 (145-2C11), anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-CD11b (M1/70), anti-B220 (RA3-6B2), anti-Gr-1 (RB6-8C5), anti-Ter119 (TER-119), anti-CD45 (30-F11), anti-FcRγII-III (93), anti-Sca-1 (E13-161.7), anti-c-Kit (2B8), anti-CD41 (MWReg30), anti-CD48 (HM-48-1), anti-CD150 (TC15-12F12.2), anti-IL7Rα (A7R34), anti-Flt3 (A2F10), anti-PDGFRα (APA5) (all from BioLegend, San Diego, CA) and anti-CD34 (RAM34; eBioscience, San Diego, CA). A mixture of CD4, CD8, CD11b, B220, Ter-119 and Gr-1 antibodies was used as the lineage (Lin) mixture. 7-AAD was used to identify and exclude dead cells. For cell cycle analysis, the cells were fixed and permeabilized using the Fixation/Permeabilization Solution kit (BD Biosciences) and were stained with PE-conjugated anti-Ki-67 antibody (BD Biosciences) and DAPI (BioLegend). The stained cells were analyzed and sorted using a FACSAria and an Accuri C6 flow cytometer (BD Biosciences), respectively. The flow cytometry data were analyzed using FlowJo ver. 10.0.5 (Treestar, Ashland, OH).

Antibodies and reagents were obtained from the following sources. PE-conjugated anti-CD31, anti-CD45, and anti-Sca-1 and APC-conjugated anti-Vcam1 antibodies were obtained from BioLegend (San Diego, CA, USA). Rabbit or mouse anti-GFP antibodies cross-reacting with YFP were obtained from Thermo Fisher Scientific (Carlsbad, CA, USA) or EMD Millipore. Mouse anti-PAX7 and mouse anti-myosin heavy chain (MF20, MAB4470) antibodies were purchased from R&D Systems (Minneapolis, MN, USA). Rabbit anti-MyoD antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-Laminin antibody was obtained from Sigma (Sigma-Aldrich, St. Louis, MO). Rat anti-Laminin α2 antibody was obtained from Enzo (Enzo Life Sciences, NY). Rabbit anti-Dystrophin antibody was obtained from Abcam (Cambridge, MA, USA). Rat anti-Ki67 antibody and DAKO Protein Block were obtained from DAKO (Tokyo, Japan). Alexa Fluor-conjugated secondary antibodies were purchased from Thermo Fisher Scientific. M.O.M. kit and mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) for nuclear staining was obtained from Vector Laboratories (Burlingame, CA, USA).

Skeletal muscles from the hind limbs were used for flow cytometric analysis. Single-cell isolation was performed as described previously [16 (link)]. In short, after removal of the fat tissues, vessels, nerves, and tendons, the muscles were minced with forceps and digested in Hanks’ balanced salt solution containing 0.2% collagenase type II (Worthington Biochemical, Lakewood, NJ, USA). The digested samples were filtered through 70- and 40-μm cell strainers to remove the debris. Red blood cells were removed using Red Blood Cell Lysis Buffer (Roche Diagnostics, Rotkreuz, Switzerland). The following fluorochrome-conjugated monoclonal antibodies were used for cell sorting: anti-CD31 (102,405, 1:50; BioLegend), anti-CD45 (103,107, 1:200; BioLegend), anti-PDGFRα (FAB1062P, 1:10; R&D Systems), anti-Sca1 (108,113, 1:80; BioLegend), anti-mouse ST2 (FAB10041A, 1:20; R&D Systems) and Brilliant Violet 421 streptavidin (405,226, 1:666; Biolegend). The biotinylated SM/C2.6 monoclonal antibody was generously provided by Dr. S. Fukada [17 (link)]. Flow cytometry was performed using the CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA, USA). Because of the easier availability of young mice, more animals were used in the analysis for young mice than for adult and aged mice.

AT-MSCs and CB-MSCs (2 × 10⁴ cells) were seeded into 6-well plates on day 0. The total number of expanded cells were counted at day 1, 3 and 5. Cell doubling time (DT) was calculated by the following formula: DT = T × ln2/ln × (Xe/Xb), where T is the incubation time in any units; Xb is the cell number at the beginning of the incubation time; Xe is the cell number at the end of the incubation time.
Cultured cells were stained with FITC-conjugated anti-CD29 or anti-c-kit, PE-conjugated anti-CD44, anti-CD45, anti-CD106 or anti-Sca-1, APC-conjugated anti-CD105, and APC-Cy7-conjugated anti-CD11b (all from Biolegend, San Diego, CA, USA) at a concentration of 0.5 μg/mL for 30 min at 4 °C. The corresponding fluorophore-conjugated isotype controls were used for the gating of the positive-stained cells. Immunophenotypic analysis of 5000–10,000 cells of each sample was performed using FACSCanto II flow cytometer (BD Biosciences). The flow cytometry data were analyzed using Flowjo software (Tree Star, Ashland, OR, USA, ver. 10.0.7). Both assays were performed three times with duplicated samples.

Primary cells obtained after the enzymatic digestion of periaorta adipose tissue or cultured cells detached with scraptase (GenDEPOT) were stained for 30 minutes at 4°C with following antibodies: anti-CD45-APC (BD Biosciences, 561018), anti-CD29-PE (BD Biosciences, 562801), anti-Sca-1 (stem cell antigen 1)-PE-Cy7 (Biolegend, 108113), anti-CD31-PerCP (Biolegend, 201419), anti-CDH5-Alexa Fluor 647 (BD Biosciences, 562242), anti-PDGFRα-APC (platelet-derived growth factor α; eBioscience, 17-1401-81), anti-CD117-PE (Biolegend, 105807), anti-CD34-APC (Biolegend, 128611), anti-CD44-PerCP (Biolegend, 103035), and anti-CD11b-PE (Biolegend, 101205). Nucleated cells were distinguished from debris with Syto16 (Molecular Probes, S7578) and dead cells with 4′,6-diamidino-2-phenylindole (DAPI). Cells were analyzed with BD Accuri C6 or BD LSR Fortessa II (both Becton Dickinson). Gating was set with appropriate fluorescence minus one controls or corresponding IgG controls.