CD31CD45SM/C-2.6+ satellite cells were sorted from WT mice and CD31CD45PDGFRα+ mesenchymal progenitors were sorted from /Bmp3b-Tg or Bmp3b-KO mice. For single culture, 2 × 103 (single) or 6 × 103 (single ×3) satellite cells were cultured per well in a 96-well CellCarrier Ultra plate (PerkinElmer). For coculture, 2 × 103 satellite cells and 4 × 103 mesenchymal progenitors were plated per well. Cells were maintained in GM at 37°C under 5% CO2 and 3% O2 for 4 days and treated with differentiation medium (DM) consisting of DMEM supplemented with 5% horse serum for 2 days at 37°C under 5% CO2 and 20% O2. The medium was replaced with fresh DM, and cells were maintained for an additional 2 days. To analyze the myotube area, cells were fixed with 4% PFA for 5 minutes followed by immunofluorescence staining of MyHC. For quantification, 6 randomly selected fields from duplicate wells were used. Images were collected and pooled from 2 independent experiments. The area of the myotube in 12 randomly selected fields was measured collectively using the WinROOF software (Mitani Corp).

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