Stempro osteogenesis supplement

Murine MSCs were stimulated for up to 2 weeks in StemPro osteocyte/chondrocyte differentiation basal medium supplemented with StemPro Osteogenesis Supplement (Life Technologies). After fixation of the cells with 4% paraformaldehyde, alkaline phosphatase activity was revealed by SigmaFast BCIP/NBT chromogen staining (Sigma Aldrich, St. Louis, MO) according to PromoCell's application note for “osteoblast differentiation and mineralization” (PromoCell GmbH, Heidelberg, Germany).

ASCs were seeded at a density of 6 × 10⁵ mL on 13 mm glass cover slips (Agar Scientific, Stanstead, UK) pre-treated with nitric acid. After 72 h, standard medium was replaced by StemPro^® Osteocyte basal medium supplemented with StemPro^® osteogenesis supplement according to manufacturer’s instructions (Life Technologies, Thermo Fisher Scientific, Loughborough, UK). Cells were cultivated for up to 21 days in a humidified incubator (Binder APT.lineTM C150, Binder, Tuttlingen, Germany) at 37 °C and 5% CO₂. Media were changed every three days. Osteogenic differentiation was assessed by Alizarin Red S staining at day 21 as described [26 (link)]. Briefly, ASCs differentiated for 21 days were fixed for 15 min using 4% PFA followed by three wash steps using PBS with 5 min per wash step. Calcium deposition was visualised by staining the cells with 1% Alizarin Red S in double-deionised water (ddH₂O, Sigma-Aldrich) at pH 4.3 for 5 min at room temperature followed by imaging using a Nikon A1R inverted confocal microscope (Nikon, Kingston upon Thames, UK).

First, primary MSCs were cultured in high-glucose DMEM (4.5 g/l) for
osteogenesis induction or in low-glucose DMEM (1 g/l) for adipogenesis
induction, both containing 10% FBS. When grown to 60–80%
confluence, the cells were added with pre-prepared adipogenic medium or
osteogenic medium. The osteogenesis or adipogenesis differentiation medium was
respectively prepared by adding 10 ml StemPro® Osteogenesis Supplement or
10 ml StemPro®Adipogenesis Supplement (Invitrogen Life Technologies) to
90-ml culture medium.