Bcip nbt alkaline phosphatase colour development kit

OCCM‐30 was seeded in 24‐well plates, cultured to reach 80% confluence, pre‐treated with 10 ng/mL TNF‐α for 30 minutes and mineralization induced for 7 days. The cells were lysed in 1% Triton X‐100 on ice for 30 minutes. The total protein concentration was dertermined using a BCA protein assay kit (Beyotime). Then, 20 µL of lysate of each well was incubated in a 96‐well plate with 100 µL of fresh solution of p‐nitrophenyl phosphate substrate at 37°C for 30 minutes. The reaction was quenched by the addition of 80 µL of 0.5 mol/L NaOH solution. The absorbance at 405 nm was measured using a microplate‐reading spectrophotometer (PowerWave XS2, BioTek). A standard curve describing the relationship between OD value and p‐NP concentration was generated with the additional examination of gradient diluted p‐NP. The produced p‐NP concentration in each sample was calculated using the standard curve, and the relative ALP activity was expressed as the percentage in produced p‐NP concentration per unit time per mg protein.
For ALP staining, the cells were seeded in six‐well plates, treated and mineralization induced as those for ALP activity analysis. ALP staining was performed using a BCIP/NBT alkaline phosphatase colour development kit (Beyotime Institute of Biotechnology). Pictures were taken using a light microscope and a camera.

After 7 days of osteogenic induction, hPDLSCs were fixed and stained according to the manufacturer's instructions of the BCIP/NBT alkaline phosphatase colour development kit (Beyotime) and the images were captured with a camera. ALP activity of hPDLSCs was measured following the manufacturer's instructions using the Alkaline Phosphatase Assay kit (Beyotime). Absorbance was evaluated spectrophotometrically at 405 nm.

After transfection with mock lentivirus, shMycn lentivirus and/or pCDH-CMV-Klf4 lentivirus, mDPCs were cultured in CM for 4 days and OM for 9 or 11 days. Cells were fixed with 4% PFA and stained with a BCIP/NBT Alkaline Phosphatase Colour Development Kit (Beyotime Biotechnology) or alizarin red assay (Sigma–Aldrich). For ALP activity quantification, cells were lysed with 0.1% Triton/PBS solution, and the ALP activity was analysed with an alkaline phosphatase assay kit (Beyotime Biotechnology). Protein concentrations were determined as mentioned above. ALP activity was defined as micromoles of reaction product (p-nitrophenol) per minute from a mg of cellular protein [25 (link)]. For ARS quantification, 10% cetylpyridinium chloride (Aladding, Shanghai, China) solution was added to the stained cells and incubated at room temperature overnight. The solutions were then collected and measured at OD 562 nm by a microplate reader.