Nbt alkaline phosphatase color development kit

The three different groups (blank, Ng/SAIB, Ng-lipo/SAIB-treated) of osteoblast cells were seeded at a density of 2 × 10⁴ cells per well in a 24-well plate, and the basal culture medium was then changed to an osteogenic-inducing medium. After 3 and 5 days of incubation, the level of ALP in the cells was measured using a commercial ALP colorimetric assay kit (Beyotime, Shanghai, China). The OD values were measured at 410 nm using an ELISA plate reader. After 7 days, the cells were stained using a 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) Alkaline Phosphatase Color Development Kit (Beyotime).

Alkaline phosphatase (ALP) staining was processed per the manufacturer’s instructions of the 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/nitro blue tetrazolium (NBT) Alkaline Phosphatase Color Development Kit (Beyotime Biotech). Human VICs were fixed in 4% PFA for 30 min at room temperature after washing in 1x PBS supplemented with Tween20 (Biosharp) (PBST). This was followed by three washes with PBST and then the cells were incubated with moderate BCIP/NBT dyeing solution at room temperature for 1 to 2 h protected from light until the desired coloration displayed. VICs were next rinsed with 1x PBS three times. Images were acquired under a microscope (CKX53, Olympus). ALP-positive areas were calculated using Image J software, and the mean was determined from three independent biological replicates.

Alkaline phosphatase staining was monitored with the 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and NBT Alkaline Phosphatase Color Development Kit (C3206, Beyotime Institute of Biotechnology) in accordance with the instructions. Briefly, cells were fixed by immersion in 4% paraformaldehyde at 4°C for 30 min and rinsed in PBS for 5 min. The alkaline phosphatase stain was then added to the plates for 30 min while protecting the plates from light. The pictures were monitored by microscopy after rinsing in deionized water for 5 min.