Alkaline phosphatase kit

Approximately 20–50 generations of cells were resuscitated and cultured to the logarithmic growth stage. Caco-2 cells were added to the AP chamber of transwell with 50-μg/mL Fuc-PM-DMEM and PM-DMEM, respectively, at the rate of 8000 cells/well. We added 1000-μL corresponding media to the BL chamber for culture, and changed the solution daily. When the Caco-2 model established on the Fuc-PM-DMEM media reached the fifth day, the Caco-2 model established on the PM-DMEM reached the seventh day, and TEER > 500 Ω ·cm², the AP and BL (AP/BL) AKP activity of the two models was detected according to the instructions of the alkaline phosphatase kit (Beyotime, Shanghai, China).

To assess the ALP activity of the supernatant-treated rBMSCs at Days 7 and 14, the co-cultured cells were washed with PBS, fixed in 4% paraformaldehyde for 20 min and stained with an Alkaline Phosphatase Kit (Beyotime Biotechnology, Shanghai, China). Optical images were taken using a Leica inverted microscope.

The samples were immersed in culture medium (1.25 cm²/mL) for 24 h and the extracts were collected for osteogenic differentiation evaluation assays. The cells (5 × 10⁴ cells/mL) were seeded in a 24-well plate and cultured with extracts from different samples supplied with 10 mM β-glycerophosphate, 100 nM dexamethasone, and 50 mM ascorbate and glutamine for 3 and 7 days. At the predetermined time, the BCIP/NBT ALP Color Development Kit (Beyotime, Shanghai, China) was used to stain ALP in the cells in line with the instructions of manufacturer. For quantitative detection, the intracellular ALP activity was quantitated using Alkaline Phosphatase Kit (Beyotime, Shanghai, China) and the total protein was measured using BCA protein quantitation kit (Themo, Waltham, MA, USA). The ALP activity was normalized with total protein content.