*2.4. Assessment of Adipogenic and Osteogenic Potencies of MSCs*

MSCs isolated from red bone marrow were grown on passage 1 in 12-well plates into an osteogenic medium (growth medium supplemented with 10 nM dexamethasone, 50 μg/mL 2-phospho-L-ascorbate, and 10 mM sodium β-glycerophosphate) at a density of 1 × 104 cells/mL or into an adipogenic medium (standard growth medium supplemented with 10 μM dexamethasone, 0.2 mM indomethacin, 1 IU/mL insulin, and 0.5 mM 3-isobutyl-1-methylxanthine) at a density of 3 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/mL. As a control, the cells maintained at the same density in a standard growth medium were used. The medium was changed twice a week; cultivation was continued for 20 days (adipogenesis) or 21 days (osteogenesis). For the analysis of adipogenic differentiation, cells were fixed with 4% formalin in 0.1 M phosphate-buffered saline, pH 7.2-7.4 (PBS), and stained with Oil Red O (Sigma, St. Louis, MO, USA) in order to detect neutral fat inclusions. For the analysis of osteogenic differentiation, the culture was fixed with a mixture of sodium citrate, acetone, and formaldehyde; cytochemical detection of alkaline phosphatase activity was carried out by the method of azo coupling of Fast Red Violet (FRV) with naphthol AS-BI (Sigma, USA), according to the manufacturer's protocol. In addition, in the culture of MSCs subjected to osteogenesis induction, deposits of calcium salts were detected by staining cells fixed with 96% ethanol with Alizarin Red S (Sigma, USA) at pH 4.1. After cytochemical reactions, the cells of all

studied cultures were additionally stained with hematoxylin and analyzed using a Primovert inverted light microscope (Zeiss, Germany).
