Alkaline phosphatase kit

A total of 5000 cells were cultured in a gelatin-coated 10cm dish. After 7–10 days, when distinct clones became visible, the ES cells were washed twice with DPBS. Subsequently, the cells were fixed with 4% paraformaldehyde for 2–5 min and stained using the Alkaline Phosphatase Kit (Sigma, St. Louis, MO, USA, cat. no. SCR004) according to the manufacturer’s instructions. The staining reaction was halted by rinsing the cells with PBS three times. The stained cells were then counted and subjected to statistical analysis.

ADSCs were conducted the osteogenic induction at passage 2 (P2) as the reported approach.23 ADSCs (1 × 10⁶ cells/well) were seeded into six‐well cell culture dishes. Then cells were cultured under the osteogenic differentiation medium for 2 weeks, namely LG‐DMEM supplemented with 1 μM dexamethasone (Sigma), 50 μM ascorbate‐2‐phosphate (Sigma), 100 μM glycerophosphate (Sigma), 10% FBS and 1% P/S. Then cells were rinsed three times with PBS, and were fixed by 4% paraformaldehyde at room temperature for 10 minutes. Then cells were rinsed three times using PBS, and stained using Alkaline Phosphatase Kit (Sigma). Lastly, cells were gently washed three times with deionized water, and were imaged under the inverted fluorescence microscope (OLYMPUS).

46C mESCs cultured on 0.1% gelatin-coated plates were washed with PBS 2–3 times and fixed in 4% paraformaldehyde. Then the Alkaline Phosphatase Kit (Sigma) was used to detect alkaline phosphatase (AP) activity.

Osteogenesis was assessed by alkaline phosphatase (ALP) assay [51] and von Kossa staining [50] (link) and by evaluating the relative abundance of the transcripts of two osteogenic differentiation markers, such as Runt-related transcription factor 2 (RUNX2) and Osteopontin (OPN). Briefly, ALP activity, a typical osteoblast marker, was assessed by using the Alkaline Phosphatase Kit (Sigma Aldrich), a commercial kit based on naphthol AS-BI and fast red violet LB [51] . After treatments in the conditions described below (Experiment 2), cells were fixed with a citrate-acetone-formaldehyde fixative for 30 sec at room temperature. After being rinsed with distilled water, cells were incubated for 15 min in dark with alkaline-dye mixture (NaNO2, FRV-Alkaline Solution, Naphthol AS-BI Alkaline Solution) and washed with distilled water. Cells were counterstained with hematoxylin solution, washed in tap water and evaluated under light microscopy. For von Kossa staining, cells were washed with PBS and then fixed with 10% formalin for 1 h at room temperature. The formalin was removed and cells were washed with distilled water. A 5% (w/v) silver nitrate solution was added and cells were exposed to UV light for 20 min. Reaction was stopped by using a 5% sodium thiosulphate solution for 2 min. Cells were washed with distilled water. Calcium phosphate deposits stained black [50] (link).

To assess the effects of CBZ on ALP activity, BMSCs were seeded into 6-well culture plates at a density of 2 × 10⁵ cells/well, and incubated with 2 mL/well OM containing different concentrations of CBZ (0, 10, 20, 40 μM) CZB for 14 days to induce differentiation. After inducting 14 days, the cell culture medium was removed and the cells were washed with PBS 2–3 times. Cells were fixed in 4% formaldehyde for 30 min, after which they were washed three times with PBS and stained by Alkaline Phosphatase Kit (MAK447, Sigma-Aldrich) under the guidance of the instructions. Then cells were observed under a microscope.