Bcip nbt alkaline phosphatase color development kit

All study protocols and animal care procedures were approved by the Animal Care and Use Committee of Fujian Medical University. Fifteen 4-week-old Sprague-Dawley rats were obtained from the animal resource center (SLAC Laboratory Animal Co, Ltd., Shanghai, China). BMSCs were isolated and cultured from rat femurs as previously described [28 (link)]. Colony formation assays were conducted and stained with 0.1% crystal violet (Sigma-Aldrich, USA). The culture medium of BMSCs was changed to osteoinductive medium (Cyagen, China) or adipoinductive medium (Cyagen, China) to confirm their multiple differentiation abilities. Alkaline phosphatase (ALP) staining was conducted using a BCIP/NBT Alkaline Phosphatase Color Development kit (Beyotime, China). Mineralization nodules were stained with Alizarin Red S (Sigma-Aldrich, USA). Lipid droplets of BMSCs were stained with Oil Red O (Sigma-Aldrich, USA). The surface markers of BMSCs were evaluated by flow cytometry. BMSCs were suspended with PBS-containing fluorescein isothiocyanate (FITC)-coupled antibodies against CD90 and CD105 (Abcam, USA). FITC-coupled nonspecific IgG (Abcam, USA) was used as an isotype control.
For the cell scaffold co-culture experiments, all scaffolds were sterilized by ethylene oxide. BMSCs were cultured on the samples placed in 24-well culture plates at an initial density of ∼ 2 × 10⁴ cells per well.

Tension group and no-tension group cells were washed by ice-cold PBS twice and fixed in 1 ml 4% paraformaldehyde for 20 min at room temperature. Alkaline phosphatase (ALP) staining was performed using BCIP/NBT Alkaline Phosphatase Color Development Kit (C3206; Beyotime, Shanghai, China) according to the manufacturer’s protocol. Then, photos were taken by a scanner (GE Image scanner III). Cellular ALP activity was detected using Alkaline Phosphatase Assay Kit (A509-2; Jian-Cheng Bioengineering Institute, Nanjing, China) and the procedure was described before (Li et al., 2020 (link)). Finally, the absorbance values related to ALP activity were recorded at 520 nm with the microtiter plate spectrophotometer (Spectrama).

BMSCs were seeded into 24-well plates and cultured until the they reached 80% confluence. Then, the medium was replaced with an osteogenesis assay kit (MUBMX-90021, Cyagen, CA, United States) with or without BAT conditioned medium (1:1) at 37°C in humidified air with 5% CO₂ for 21 days to induce osteogenesis. Bone formation was detected using alkaline phosphatase (ALP) or alizarin red staining on days 14 and 21. ALP staining was performed as follows: after washing three times with PBS and fixation in 4% paraformaldehyde for 10 min at room temperature, cultured cells were stained using the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime Institute of Biotechnology, Shanghai, China). All steps were strictly in accordance with the manufacturer’s instructions. After 21 days of culture, alizarin red staining was performed. Briefly, the cultured cells were washed with PBS and fixed in 4% paraformaldehyde for 30 min, and then 500 µL alizarin red dye (contained in the MUBMX-90021 kit) was added to each well and incubated at room temperature for 10 min. After washing five times with PBS, 10% cetylpyridinium chloride (500 µL) (H811089, Macklin, CA, United States) was added to each well for semiquantitative analysis. Then, the absorbance of the supernatant at 562 nm was detected after incubation for 30 min at room temperature.

The cells were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde at room temperature for 30 min, the residual liquid was removed and rinsed with PBS.
ALP staining was performed according to the instructions of BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China) and incubate cells. ARS staining was performed by incubating cells with 0.02% ARS. All were carried out at room temperature. When the coloration reaches the desired depth, the reaction was terminated by removing the residual liquid and washing twice with PBS. Finally, the color depth was observed under the microscope and photographed. Quantitative analysis of ALP staining and ARS staining was performed using Image pro plus.

BMSCs were plated in 6-well plates at a density of 2 × 10⁵ cells per well for 12 h and incubated for 7 days and 14 days under the same treatment conditions as above, and the solution was changed every 3 days. After 7 days, the cells were collected and lysed, and the total protein amount of each group was determined using the BCA kit (Beyotime, Shanghai, China). After verifying that the total protein amount of the ordinary exosome group, the inflammatory exosome group, the CM group, and the CM+GW4869 group were all at the same level, the alkaline phosphatase activity of each group was detected using the ALP kit (Built, Nanjing, China). Each experiment was repeated 3 times.
After 14 days, a BCIP/NBT alkaline phosphatase color development kit (Beyotime, Shanghai, China) was used according to the provided directions. The cells were washed three times with PBS and fixed with 4% paraformaldehyde for 30 min, then treated with BCIP/NBT substrate for 10 h, A microscope was used to analyse the colorimetric changes and a scanner was used to image the stained cells. Absorbance was then measured at 450 nm. Experiments were repeated in triplicate.