Bcip nbt alp colour development kit

At the end of the iPTH treatment, the ALP activity in osteoblasts was measured. A BCIP/NBT ALP colour development kit (Beyotime, China) was used for staining and an ALP assay kit (Beyotime, China) was used for the quantitative assay according to the manufacturer’s instructions.

For investigating the extent of osteogenic differentiation in the 2D and 3D environment, BMSCs with and without hydrogel were cultured in osteogenic medium without exosomes for a fixed time interval. To measure ALP expression, cells were fixed with 10% formalin, stained using BCIP/NBT ALP Colour Development Kit (Beyotime), and imaged with an inverted fluorescence microscope (DMI6000 B, Leica, Germany). To quantitate ALP activity, cells were tested with an ALP Assay Kit (Beyotime). Absorbance was measured at 405 nm. To investigate mineral deposition, cells were fixed with formalin and stained with Alizarin Red S Staining Kit for Osteogenesis (Beyotime) for approximately 20 min, washed three times with dH₂O and stained with ARS for 20 min at room temperature. Cells were imaged with a Leica MDI6000 B fluorescence microscope. After several washes with dH₂O, the stain was desorbed with 200 µL 10% cetylpyridinium chloride (Sigma, Germany) for 1 h. The dye was collected, and the absorbance was read at 590 nm using a spectrophotometer.

ALP staining and ALP activity assays were performed using BMSCs that had been cultured in OIM for 3 days. For ALP staining, BMSCs were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 min. Next, the cells were washed three times with PBS and stained using a BCIP/NBT ALP Colour Development Kit (Beyotime, Shanghai, China) following the manufacturer’s instructions. For measurements of ALP activity, BMSCs were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich, MO, USA). The ALP activity in the cellular fraction was measured using a microplate test kit (Nanjing Jiancheng Biotechnology Co Ltd., Jiangsu, China) following the manufacturer’s instructions, and the absorbance at 520 nm was measured using a microplate reader.

To induce osteogenic differentiation, 5 × 10⁴ cells were inoculated into each well of the 12 well plate. Added 50 μg/mL ascorbic acid and 10 mM of β-glycerol phosphate medium was used as osteogenic medium and was set as OB group.
After 7 days of osteogenesis induction, osteoblasts were stained with BCIP/NBT ALP Colour Development Kit (Beyotime Biotech, China) according to the manufacturer’s plan. The activity of ALP was measured by the absorbance of ALP Assay Kit (Nanjing Jiancheng Bioengineering Institute, China).

TDSCs seeded into 12-well plates were induced with an osteogenic medium using different concentrations of GANT58 and SAG for 7 days, fixed in 4% PFA for 30 min, and then stained with a BCIP/NBT ALP colour development kit (Beyotime, C3206) as instructed. The positive rates of ALP staining were quantified on ImageJ.

ALP activity was qualitatively and quantitatively evaluated using commercially available kits. In the qualitative assay, MSCs at a density of 2 × 10⁴ cells were grown on glass samples with and without NTS and cultured in osteogenic differentiation media (Gibco, REF#: A10069-01). After 3 and 7 days of co-cultivation, the cells on the specimens were washed with phosphate buffer saline (PBS), fixed with 4% paraformaldehyde and stained with BCIP/NBT ALP colour development kit (Beyotime Biotech, Songjiang, Shanghai, China).
In the quantitative assay, the cells cultured on the samples were rinsed with PBS and lysed in lysis buffer containing 0.1% Triton X-100 (Sigma Chemical Company, St. Louis, MO, USA). After 2 min of centrifugation, the supernatant was used in measuring ALP activity with an alkaline phosphatase assay kit according to the manufacturer’s protocol (Beyotime Biotech, Songjiang, Shanghai, China). The measurement was based on the conversion of colourless p-nitrophenyl phosphate to coloured p-nitrophenol after co-incubation for 30 min at 37 °C. The results were normalised to the total intracellular protein content determined with a bicinchoninic acid protein assay kit (Beyotime Biotech, Songjiang, Shanghai, China). The total intracellular protein content was expressed in nanomoles of produced p-nitrophenol per minute per milligram of protein (nmol/min/mg protein).