Alkaline phosphatase detection kit

In all, 1 × 10³ single E14 mESCs of different conditions were seeded in a 6-well plate and 1 or 2 × 10³ hESCs were plated into a 6 cm dish. Ten days after seeding mESCs and 14 or 18 days for hESCs, alkaline phosphatase staining was performed with an alkaline phosphatase detection kit (Beyotime) according to the manufacturer’s instructions. The number of colonies was then quantified after the staining.

Alkaline phosphatase (AP) staining was performed using the Alkaline Phosphatase Detection Kit (Beyotime, Shanghai, China).

Alkaline phosphatase activity was determined using the alkaline phosphatase detection kit (Beyotime, China) following the manufacturer’s protocol. Briefly, cells were lysed with cell lysis buffer (not containing phosphatase inhibitors), and the supernatant was centrifuged for alkaline phosphatase detection. The absorbance of the supernatant at 405 nm was measured using a spectrophotometer (Bio-Rad). The alkaline phosphatase activity of the samples was calculated based on the absorbance of the blank control, standard, and sample.

In all, 2 × 10⁴ single ESCs were seeded per well in 24-well plate and were grown for 3 days. The alkaline phosphatase detection kit (Beyotime, China, Cat#C3206) was used for this assay according to the instruction. Briefly, cells were fixed with 4% paraformaldehyde at room temperature for 20 min, followed by incubated with staining mixture in dark overnight at room temperature. Colonies were visualized and photographed using microscopy. AP staining was also used to measure the numbers of pluripotent colonies in colony-forming assay.

ALP staining and quantitative analysis: The ALP staining assay was performed in examination of osteogenesis activity of different groups. The cells in the medium of a specific group were cultured for 7 d and stained according to the instructions of the ALP staining kit (Beyotime, Shanghai, China) after incubation of 1% Triton X-100 for 10 min (min). Quantitative assay was performed using alkaline phosphatase detection kit (Beyotime, Shanghai, China). Para-nitrophenol(p-nitrophenol) can be generated from para-nitrophenyl phosphate under alkaline phosphatase for determination of absorbance at 400 to 415 nm.
ARS and quantitative analysis: The alizarin red was performed in the examination of the differentiation of hBMSCs by aggregating with calcium to form chelates. The same cultured cells like ALP staining in the proper plate with medium according to specific groups for 14 d. Then, the experimental cells were rinsed by ultrapure water 3 times and then incubated with 4% paraformaldehyde for 15 min. After fixing and washing, the cells were incubated with alizarin red for 30 min and washed with sterile water. For quantitative analysis, alizarin red was dissolved with 10% cetylpyridinium chloride, and then the lysate was detected at 562 nm with the microplate reader.

P19 cells were placed onto 6-well plates at a density of 1000 cells/well, and gently shaken to make them evenly distributed in the plate without aggregation. Then, the fresh culture medium containing the compounds was replaced every day. After one week, the cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature. Subsequently, the fixed cells were washed twice with PBS and then stained using the Alkaline Phosphatase Detection Kit (Beyotime Biotechnology, China) following protocols provided by the manufacturer.