Stemxvivo human osteogenic supplement

The ability of culture-expanded MSCs to differentiate into the osteogenic lineage was validated according to earlier described methods (Herberg et al., 2013 ; Gregory et al., 2004 (link)). In brief, cells were plated in 12-well plates at 50000 cells/cm² and cultured in DMEM for 24 h. Culture medium was then aspirated and replaced with StemXVivo Osteogenic/Adipogenic Base Media (#CCM007 R&D Systems) supplemented with StemXVivo Human Osteogenic Supplement (#CCM008 R&D Systems). Treatment-containing medium was replaced 2 times per week. The early osteogenic differentiation marker, Alkaline Phosphatase, was assessed in cell culture media after 7 days using an Alkaline Phosphatase Assay Kit (#ab83369 Abcam). After 3 weeks, osteogenic differentiation was assessed by staining with Alizarin-Red Staining Solution; (#TMS-008-C Millipore Sigma). The cells were fixed with 10% formalin for 20 min at room temperature (RT) and stained with Alizarin-Red Staining Solution for 20 min at RT. Stained monolayers were visualized by phase-contrast microscopy using an inverted microscope (Nikon, Melville, NY). Differentiation was quantified as previously described (Ripoll and Bunnell, 2009 (link)). In brief, cells were destained using 10% cetylpyridinium chloride (#855561 Sigma-Aldrich) and collected samples analyzed using a microplate reader at 570 nm.

To determine whether KYN affected BMSCs density, we utilized a Crystal violet Assay Kit (#ab232855 Abcam) according to the manufacturer's protocol. In brief, BMSCs were plated in 96 well plates at 5000 cells/well and cultured in DMEM for 24 h. Culture medium was then aspirated and replaced with StemXVivo Osteogenic/Adipogenic Base Media (#CCM007 R&D Systems) supplemented with StemXVivo Human Osteogenic Supplement (#CCM008 R&D Systems) with or without different doses of KYN (10, 50, 200 μM). After 3 days, the culture media was removed and the cells were washed and stained with the Crystal Violet Staining solution for 20 min at RT. Then, the staining solution was removed and the remaining stain was solubilized for 20 min with the Solubilization Solution. Finally, the Crystal Violet stain was quantified using a microplate reader at 595 nm.