Osteogenic medium

For osteogenic differentiation, passage 2 AT-MSCs were seeded on 6-well plates (5.0 x 10³ cells/cm²) and incubated in H-DMEM supplemented with 10% FBS and 1% antibiotic–antimycotic solution for 24 h. The medium was then changed to osteogenic medium (Cell Applications, San Diego, CA) [18 (link)]. The medium was changed twice-weekly. For osteogenic analysis, mineral deposits were quantitatively analysed by von Kossa staining after 21 days.
For adipogenic differentiation, passage 2 AT-MSCs were seeded on 6-well plates (8 x 10³ cells/cm²). The cells were cultured in H-DMEM supplemented with 10% FBS and 1% antibiotic–antimycotic solution until they reached confluence, and then the medium was changed to canine adipocyte differentiation medium (Cell Applications, San Diego, CA) [18 (link)]. The medium was changed twice-weekly. Adipogenesis was analysed by Oil Red O staining after 21 days.

For osteogenic differentiation, ADSCs and LDSCs at passages 2, 4, and 6 were seeded in 6-well plates (5.0 × 10³ cells/cm²) and incubated in H-DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic solution for 24 h. The medium was then changed to osteogenic medium (Cell Applications) [13 (link)]. The medium was changed twice weekly. For osteogenic analysis, mineral deposits were quantitatively analyzed by von Kossa staining after 21 days.
For adipogenic differentiation, ADSCs and LDSCs at passages 2, 4, and 6 were seeded in 6-well plates (8 × 10³ cells/cm²) and cultured in H-DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic solution until confluency. The medium was changed to canine adipocyte differentiation medium (Cell Applications) [13 (link)]. The medium was changed twice weekly. Adipogenesis was analyzed by Oil Red O staining after 21 days.
Positive-stained areas were measured with ImageJ. Four fields were randomly selected from culture dishes divided equally into four regions.

For osteogenic differentiation, passage 2 AT-MSCs were seeded on 6-well plates (5.0 × 10³ cells/cm²) and incubated in H-DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic solution for 24 h. The medium was then changed to osteogenic medium (Cell Applications) [10 (link)]. The medium was changed twice weekly. For osteogenic analysis, mineral deposits were quantitatively analyzed by von Kossa staining after 21 days.
For adipogenic differentiation, passage 2 AT-MSCs were seeded on 6-well plates (8 × 10³ cells/cm²) and cultured in H-DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic solution until confluency. Then, the medium was changed to canine adipocyte differentiation medium (Cell Applications). The medium was changed twice weekly. Adipogenesis was analyzed by Oil Red O staining after 21 days.