Stempro osteogenic induction medium

MSC were seeded at 600 cells per well in 384-well plate in MSC culture medium and treated as previously described. After 72 h, MSC medium was replaced by STEMPRO osteogenic induction medium (Invitrogen) in the presence or not of the different drugs. After 7 days of treatment, cells were fixed with ethanol 95% and stained by adding either a colorimetric substrate of the alkaline phosphatase, the 5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma-Aldrich) or a chromogenic substrate of this enzyme (absorbance at 405 nm), the p-nitrophenyl phosphate (Pierce Biotechnology, Rockford, IL, USA).

Cells were seeded at 12,000 cells for WT MSC and 19,000 cells for HGPS MSCs per well in 24-well plates in MSCs culture medium and treated as previously described. After 72 hours, the MSC medium was replaced with STEMPRO osteogenic induction medium (Invitrogen), in the presence of or without the different drugs. After 7 days of treatment, the cells were fixed with 95% ethanol and stained by adding either a colorimetric substrate for alkaline phosphatase, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (NBT) (Sigma-Aldrich), or a chromogenic substrate for this enzyme (absorbance at 405 nm), p-nitrophenylphosphate (pNPP) (Pierce Biotechnology). Cell viability was measured by counting Hoechst stained cells using a LEAP cell processing workstation (Cyntellect). Data are expressed as percentages relative to the control values which are defined as 100%.

The cells were seeded at 12,000 cells per well for WT MSCs and 19,000 cells per well for HGPS MSCs in 24-well plates in MSC culture medium and treated as previously described. After 72 h, the MSC medium was replaced by STEMPRO osteogenic induction medium (Invitrogen) in the presence or absence of the different drugs. After 7 days of treatment, the cells were fixed with ethanol 95% and stained by adding either a colorimetric substrate of alkaline phosphatase, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma-Aldrich, St Louis, MO, USA), or a chromogenic substrate of this enzyme (absorbance at 405 nm), p-nitrophenyl phosphate (Pierce Biotechnology, Rockford, IL, USA). The cell viability was measured using a luminescent assay, CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA), based on ATP quantification. The reagent was added to the cell medium and, after 30 min of incubation, the luminescent signal was quantified using an Analyst GT counter luminometer (Molecular Devices, Sunnyvale, CA, USA).