Goat serum

Cells were fixed in 4% paraformaldehyde for 10 min and treated with 0.1% Triton X-100 for 10 min at RT. After blocking with 5% goat serum (Cedarlane)/2% bovine serum albumin (BSA; Sigma) in PBS, the cells were reacted with primary antibodies: a rabbit polyclonal myogenin antibody (Santa Cruz Biotechnology) and an anti-pan myosin heavy chain (MHC) antibody, MF20 (R&D Systems) at a 1:200 dilution overnight at 4 °C. To detect the differentiation of MSCs, an anti-human osteocalcin antibody (R&D Systems, #967801), an anti-mouse FABP4 antibody (R&D Systems, #967799), or an anti-human aggrecan antibody (R&D Systems, #967800) was used. For detection of undifferentiated human iPS cells, an anti-hSOX2 antibody (Cell Signaling, #3579), anti-hNANOG antibody (R&D Systems, AF1997), or an anti-hOCT-3/4 antibody (R&D Systems, AF1759) was used. The next day, after washing with PBS (−), the cells were incubated with Alexa 568 goat anti-mouse IgG2b, Alexa 488 goat anti-rabbit IgG, Alexa 568 donkey anti-goat IgG, or Alexa 488 donkey anti-mouse IgG (Molecular Probes) for 2 h. The nuclei were then stained with DAPI (Tokyo Chemical Industry). The images were recorded with an all-in-one microscope (BZ-X810) and analyzed with hybrid cell count software (Keyence).

Myofibers were fixed in 4% paraformaldehyde (PFA) in PBS and 1% glycine and blocked in PBS containing 0.2% Triton X-100 (BioShop), 2% BSA, 5% goat serum (Cedarlane), and 1% azide. Myoblasts were fixed in 2% PFA in PBS and blocked in PBS containing 0.3% Triton X-100 and 10% goat serum. Cryosections were thawed at room temperature, fixed in 4% PFA, and processed for antigen retrieval in citrate buffer at 95 °C for 20 min. The sections were permeabilized with PBS containing 0.5% Triton X-100 and blocked in PBS containing 0.1% Triton X-100 and 5% donkey serum (Cedarlane) prior to incubation with primary antibody overnight at 4 °C. The cells were washed with PBS and incubated in biotin anti-mouse (when indicated) or secondary antibodies conjugated to a fluorescent dye (Cy3, Alexa 488, or Alexa Flour 647; all from Jackson ImmunoResearch). Nuclei were counterstained with DAPI (0.5 μg/ml). The primary antibodies used were as follows: Pax7-c (DSHB), MYH (H-300; Santa Cruz), MyoD (C-20; Santa Cruz), myogenin (M-225; Santa Cruz), C/EBPβ (E299; Abcam), and laminin (AL-4; Millipore).

After FACS sorting, 1.0 × 10⁴ Calcein^low, Calcein^middle, and Calcein^high cSCs, respectively, 4 days after isolation were seeded onto 24-well plates and the numbers of cells per field were counted every day. For BrdU assay, cSCs 4 days after isolation were treated with 10 μM BrdU (Sigma Aldrich) in DMEM (high glucose, sodium pyruvate, and GlutaMAX supplement) supplemented with 20% FBS, 1% CEE, and 1% PS at 37 °C with 5% CO2 for 2 h. The cells were then treated with 50 nM calcein-AM followed by FACS sorting. Sorted Calcein^low, Calcein^middle, and Calcein^high cSCs were fixed with 4% paraformaldehyde/phosphate-buffered saline (PBS). After washing, fixed cells were treated with 0.1% Triton X-100/PBS for 10 min for permeabilization, followed by 2 N HCl for 30 min at room temperature, blocking with 5% goat serum (Cedarlane) in 2% bovine serum albumin (BSA)/PBS for 15 min, and incubation with anti-BrdU (1:400, clone: BU1/75 (ICR1); AbD Serotec) in 2% BSA/PBS at 4 °C overnight. After washing, the cells were incubated with Alexa Fluor 488-labelled secondary antibody (1:1000; Thermo Fisher Scientific) in 1% BSA/PBS. After several washings, nuclei were stained with DAPI (Dojindo). Immunofluorescent-staining images were evaluated by fluorescence microscopy (Olympus IX71), and the percentage of BrdU-positive cells was counted.

At 24 h after transfection, transfected cells were incubated with FACS buffer (DPBS containing 0.1 % BSA and 0.1 % sodium azide) containing 10 % goat serum (Cedarlane, Ontario, Canada) on ice. After incubation, the cells were washed twice with FACS buffer and labeled with anti-HA polyclonal antibody and anti-FLAG M2 monoclonal antibody for 30 min on ice. After washing, anti-mouse IgG fluorescein (FITC) labeled secondary antibody (American Qualex, San Clemente, CA) and anti-rabbit IgG phycoerythrin (PE)-labeled secondary antibody (Biolegend, San Diego, CA, USA) was added and the cells were further incubated for 30 min on ice. Stained cells were then analyzed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA).

HCEP cells were seeded onto Lab-Tek8 well-chamber slides (ThermoSci., Logan, UT, USA) using honey supplemented medium. Cells were fixed with 4% paraformaldehyde at 4°C for 10 min and then permeabilised with phosphate buffered saline containing 0.1% Triton-X and 0.1% Tween-20 (all from Sigma, St Louis, MO, USA). Cell samples were washed with PBS and blocked with 10% goat serum (Cedarlane Lab, Ontario, Canada) for 1 h. The cells then were incubated with rabbit anti-p63 (Santa Cruz Biotechnology, Heidelberg, Germany) and mouse anti-keratin 3 (Clone AE5, Millipore, Billerica, MA, USA) primary antibodies overnight at 4°C. Samples were washed thrice with PBS and incubated with AlexaFluor488 goat anti-rabbit secondary antibody at 37°C for 2 h. Samples were then counterstained with DAPI and imaged using a confocal microscope (Olympus, Japan).