Bcip nbt alp kit

BMCSs were seeded at a density of about 2 × 10⁴ cells/ml and cultured in osteogenic induction media after adhering supplemented with 100 μg/ml ascorbic acid, 2 mmol/L β-glycerophosphate, and 10 nmol/L dexamethasone. Alkaline phosphatase (ALP) staining was conducted using a BCIP/NBT ALP kit (Beyotime, Shanghai, China) on days 4 and 7 of the cell culture following the manufacturer’s instructions.

For alkaline phosphatase (ALP) staining, MSCs were washed thoroughly with PBS, fixed with 4% paraformaldehyde for 30 min and stained using a BCIP/NBT ALP kit (Beyotime, C3206) according to the manufacturer’s instructions. For the ALP activity assay, ALP activity kits (Nanjing Jiancheng Biotech, A059-2-2) were used in accordance with the manufacturer’s instructions. Briefly, MSCs were lysed using RIPA buffer containing 1% PMSF and phosphatase inhibitors. The lysates were centrifuged at 14,000 rpm and 4 °C for 30 min, and the supernatants were incubated with reaction buffer at 37 °C for 15 min. The absorbance was measured at 405 nm after adding a stop solution. The total protein concentration was detected with a BCA assay kit, and ALP activity is shown as units per gram protein per 15 min (U/g pro/15 min).

Osteogenesis of hPDLSCs was induced by an osteogenic induction medium. After incubation for 7 d, the expression of alkaline phosphatase (ALP) of hPDLSCs was determined using the ALP assay kit (Beyotime). After 14 and 21 d of culture, hPDLSCs were treated with a BCIP/NBT ALP Kit (Beyotime) for 15 min or alizarin red S (ARS) staining Kit (Beyotime) for 30 min. Finally, images were visualized under a microscope (Leica, Germany).

5 × 10⁴ hPDLFs were seeded into 12-well plates. When the density reached 70%, the hPDLFs were cultured in osteogenic differentiation medium for 7 days or 21 days, and the hPDLFs were treated in different groups every 2 days. Cells were fixed with 4% PFA (Biosharp, Beijing, China) and stained with BCIP/NBT ALP kit (Beyotime, Shanghai, China) after 7 days of medication. After 21 days of medication, cells were fixed with 4% PFA and stained with 2% Alizarin Red (Solaribo, Beijing, China). Quantify and process data using ImageJ. Each group was set up with 3 duplicate holes, and each experiment was repeated three times.

The third-passage ASCs were seeded into 6-well plates at a density of 5 × 10⁴ cells/ml. After culturing for 24 hours, GM was replaced by osteogenic differentiation medium (OM), which included GM supplemented with ascorbate (5 μmol/l), β-glycerophosphate (10 mmol/l), and dexamethasone (100 nmol/l), as well as 40 μg/ml AGEs along with various concentrations of irisin according to different groups. During the osteogenic differentiation period, OM with different concentrations of irisin along with 40 μg/ml AGEs were changed every other two days. A BCIP/NBT ALP kit (Beyotime, China) was used to detect ALP on day 7 of osteogenic induction. ARS was carried out after 21 days of osteogenic induction in vitro. Images of stained cells were acquired under an inverted phase-contrast microscope. Stained mineralized nodules were dissolved with 10% cetylpyridinium chloride (Sigma-Aldrich, USA), and the OD value was measured at 570 nm.

A BCIP/NBT ALP kit (Beyotime) was used to stain BMMSCs after 7 days of osteogenic incubation. An alkaline phosphatase assay kit (Nanjing Jiancheng Bioengineering) was used to evaluate ALP activity. BMMSCs were fixed with 4% paraformaldehyde and stained with 2% Alizarin Red S following 4 weeks of osteogenic induction. The calcium nodules were dissolved in 10% cetylpyridinium chloride solution for 1 h (Rhawn) as previously reported,
⁹ (link) and the absorbance was measured at 560 nm.