β glycerol phosphoric acid

The basic differentiation potential of ERCs and IL-1β-primed ERCs was measured. Briefly, the third generation of ERCs and IL-1β-primed ERCs (3 × 10⁵ cells) were inoculated in a 6-well plate. When the confluence of cells reached more than 80%, the medium was changed with a pre-configured adipogenic medium, which was constructed of DMEM, 10% FBS, 10⁻⁶ mol/L dexamethasone, 10 μg/mL insulin, 0.5 mmol/L isobutyl methylxanthine, and 60 μmol/L indomethacin (Sigma-Aldrich, USA). After the culture for 2 weeks, cells were harvested and fixed with 4% paraformaldehyde for 30 min and washed three times with PBS. Oil Red was applied to stain cytoplasmic fat.
Additionally, the above third-generation cells were also inoculated with the osteogenic medium, which contains 10⁻⁸ mol/L dexamethasone, 10 mmol/L β-glycerol phosphoric acid, and 100 mmol/L ascorbic acid (Sigma-Aldrich). On the 10th day, cells were washed with PBS and fixed with 4% paraformaldehyde. Following which, alkaline phosphatase solution was applied to staining for 30 min to evaluate the osteogenic capacity. Purple staining cued the synthesis of alkaline phosphatases by osteoblasts.

MB-MSCs were seeded at 4 × 10⁵ cells/well in a 6-well chamber slide with 3 mL media per well. After cells adhered overnight, the medium was replaced with osteogenic medium containing DMEM-F12, 10% FBS, 10⁻⁸ mol/L dexamethasone, 10 mmol/L β-glycerol phosphoric acid, and 100 mmol/L ascorbic acid (Sigma-Aldrich St. Louis, MO, USA), and cultures were continued for another 7 days.
The osteogenic capacity of MB-MSCs was evaluated by ALP staining. Cells were fixed in 4% paraformaldehyde for 30 s at room temperature, washed with water three times, and then incubated for 30 min with staining solution, which was prepared by dissolving Fast Blue RR Salt (Sigma-Aldrich, St. Louis, MO, USA) in 48 mL of ddH₂O and 2 mL of Naphthol AS-MX Phosphate Alkaline (Sigma-Aldrich, St. Louis, MO, USA). Then, cells were washed with PBS and incubated for 30 min with Mayer's Hematoxylin (Sigma-Aldrich, St. Louis, MO, USA). The stained cells were washed and imaged under microscope. Purple staining indicated the synthesis of ALP by osteoblasts.