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 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.
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