MC3T3-E1 cells and pOBs were plated in 24-well plates in BM and when cells reached confluence were cultured in osteogenic medium in DMEM, as above, supplemented with 50 µM ascorbic acid, 10 mM β-glycerophosphate and 100 nM dexamethasone (Sigma-Aldrich), for 14 days (for the MC3T3-E1 cells) or 7 days (for pOBs). Cells were induced to mineralize in G or G+PA25 or G+PA25+Mdivi-1 treatment conditions. The culture medium with supplementation was replaced every 2 days.
Cells were fixed for Alkaline Phosphatase staining with the Leukocyte Alkaline Phosphatase kit (Sigma-Aldrich) following the manufacturer’s instruction, or for the evaluation of calcium mineralization by Alizarin Red S (ARS) staining and quantification according to standard procedures, reading absorbance at λ405 nm [4 (link)]. ARS data are represented as fold change relative to G condition. ALP+ and ARS stained OBs were photographed using an inverted microscope Leica DMi1 (Leica Microsystems, Wetzlar, Germany). Alkaline Phosphatase activity (ALP activity) was also evaluated by the Alkaline Phosphatase Activity Assay Kit (Elabscience, Houston, TX, USA), according to the manufacturer’s protocol and calculated as enzymatic units/mg protein.
Cells were fixed for Alkaline Phosphatase staining with the Leukocyte Alkaline Phosphatase kit (Sigma-Aldrich) following the manufacturer’s instruction, or for the evaluation of calcium mineralization by Alizarin Red S (ARS) staining and quantification according to standard procedures, reading absorbance at λ405 nm [4 (link)]. ARS data are represented as fold change relative to G condition. ALP+ and ARS stained OBs were photographed using an inverted microscope Leica DMi1 (Leica Microsystems, Wetzlar, Germany). Alkaline Phosphatase activity (ALP activity) was also evaluated by the Alkaline Phosphatase Activity Assay Kit (Elabscience, Houston, TX, USA), according to the manufacturer’s protocol and calculated as enzymatic units/mg protein.
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