Bcip nbt solution

Dental MSCs were cultured in a osteogenic medium containing 100 μmol/L ascorbic acid, 2 mmol/L b-glycerophosphate, and 10 nmol/L dexamethasone. After induction, cells were washed with PBS and fixed with 4% paraformaldehyde and incubated in BCIP/NBT solution (Beyotime, Shanghai, China) in the dark. Areas that stained purple were regarded as positive. ALP activities were determined using p-nitropheyl phosphate (Sigma) as described previously [24 (link)].

For ALP staining, after induction for 14 days, cells were fixed with 95% ethanol and stained with BCIP/NBT solution according to the manufacturer's protocol (Beyotime Institute of Biotechnology) at room temperature for 2 h. The ALP-positive cells were stained blue/purple. Stained cells were visualized using the Canon IXUS210 camera (Canon, Inc.; magnification). In addition, an Alkaline Phosphatase Staining Kit (BestBio, Inc.) was used to detect the ALP activity, according to the manufacturer's protocol.
For alizarin red staining, after induction for 14 days, cells were washed one or two times with phosphate-buffered saline (PBS), fixed with 95% ethanol for 10 min, washed one or two times with PBS again, covered, and stained with 0.1% alizarin red solution for 10 min. Finally, they were rinsed with PBS and observed under an inverted light microscope. For quantification analysis, 10% hexadecyl pyridinium chloride monohydrate (CPC; Sigma-Aldrich; Merck KGaA) was used to dissolve the mineralized nodules and then the absorbance was measured at 540 nm using a Multiskan™ FC spectrophotometer (Thermo Fisher Scientific, Inc.).

ALP activity was determined using an ALP kit (Sigma-Aldrich) according to the manufacturer's instructions. Cells were washed twice with 0.1 M DPBS, and lysed in 10 mM Tris–HCl containing 2 mM MgCl₂ and 0.05% Triton X-100 (pH 8.2) at 4°C. The lysates were centrifuged at 12,000 g for 10 min at 4°C, and the supernatants were mixed with ALP detection buffer and incubated at 37°C for 30 min. The reaction was then stopped by the addition of 0.1 M NaOH and monitored at 405 nm. Total protein was measured spectrophotometrically using a Micro-BCA Protein Assay Kit (Pierce, Rockford, IL, U.S.) and read at 562 nm. The enzymatic activity of ALP was normalized to the total protein content of the sample (405/562 nm) [29] (link). For ALP staining, cells were fixed with 4% paraformaldehyde for 30 min at room temperature (RT), washed with 0.1 M DPBS, and stained with BCIP/NBT solution (Beyotime) for 30 min at RT in the dark. The alkaline-phosphatase-positive cells were stained blue and purple.

ALP activity was detected after 7 days of osteogenic induction. The cells were rinsed with PBS and fixed with 4% paraformaldehyde in PBS for 15 min. And 800 μL BCIP/NBT solution (Beyotime) was added to 6-well plates for 1 h. The chromogenic reaction reflects ALP activity, which was quantified by alkaline phosphatase assay kit (Nanjing Jiancheng Biological Engineering Institute, China) following the manufacturer’s instruction.
Calcium deposition was detected after osteogenic induction for 14 days. The cells were rinsed with PBS and fixed with 4% paraformaldehyde in PBS for 15 min. After rinsing with PBS, cells in 6-well plates were stained with 1% Alizarin red S (Sigma) for 30 min. The chromogenic reaction reflects calcium deposition. Calcium content was quantified by calcium detection kit (Nanjing Jiancheng Biological Engineering Institute) following the manufacturer’s instruction.

Cells were washed twice with PBS and fixed with 4% paraformaldehyde at the room temperature for 10 min, followed by three additional ddH₂O washes. Then, fixed cells were added with BCIP/NBT solution (Beyotime, Shanghai, China), following the instructions. Stained cell photos were taken using a digital camera.