Alp activity assay kit

Differentiation of hBMSCs was evaluated using alkaline phosphatase (ALP) staining, ALP activity detection, and alizarin red staining (ARS). ALP staining was performed as previously described (Wu et al., 2012 (link)) using an ALP staining kit (Beyotime, Shanghai, China). ALP activity was measured using an ALP Activity Assay Kit (Elabscience, Wuhan, China). ARS was performed using an osteoblast mineralized nodule staining kit (Beyotime, Shanghai, China). The detailed steps are provided in the Supplementary Material.

To investigate osteogenic differentiation, BMSCs were cultured on DE-FS (n = 6 each group) loaded with or without exosomes supplemented with osteogenic media for 2 weeks. Then, the cells underwent 2 times of PBS washing, followed by 4% paraformaldehyde fixation for 5 min, and incubation with 2% Alizarin Red S (ARS) solution (Sigma) for 30 min. Finally imaged using a microscope (Nikon, Tokyo, Japan). To quantify the calcium amount on the DE-FS, the red matrix precipitate was solubilized in 10% cetylpyridinium chloride (Sigma). The eluted stain was read at 562 nm. In addition, alkaline phosphatase (ALP) activity of BMSCs that cultured on DE-FS (n = 6) loaded with or without exosomes undergo differentiation by incubation in osteogenic medium for 7 days was detected using an ALP Activity Assay Kit (Elabscience, Wuhan, China). The wavelength of 520 nm was set for detection in microplate reader. The data was normalized to the total protein contents determined by a BCA Protein Colorimetric Assay Kit (Elabscience).

Mouse serum levels of calcium were measured on an automatic biochemistry analyzer (Rayto Chemray-800, China). ALP activity was analyzed by a p-nitrophenyl phosphate (PNPP) method using an ALP Activity Assay kit (Elabscience, China) as per the manufacture's protocol. Briefly, mouse serum (5 μL) was incubated with ALP reagent (50 µL) at 37 ℃ for 15 min. After adding the detecting reagent, the absorbance was measured at 520 nm and compared to a standard curve to calculate the ALP activity (U/100 mL).