Labassay alp colorimetric assay kit

To identify the optimal concentration of icariin, cell proliferation assay and alkaline phosphatase (ALP) activities were performed with a time-course and dose-dependent setup. Cell proliferation was assessed using the cell counting kit-8 (CCK-8) (Yeasen, Shanghai, China). 2000 BMSCs per well were seeded in 96-well plates. After refreshing the medium 24 h after seeding, 100 μL of culture medium with different concentrations (0, 10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶, 10⁻⁵, and 10⁻⁴ Mol/L) of icariin (Tauto Biotech, Shanghai, China) was added to each well. Six wells per group were measured. The treatment medium was refreshed every 3 days. After 1-, 3-, 5-, and 7-day incubation, the treatment medium was replaced with 100 μL CCK-8 working solution according to the manufacture's instruction. After a 40 min incubation, the OD (optical density) values were measured at 450 nm. ALP activity and total protein content were measured after the treatment for 1 day, 3 days, 5 days, and 7 days. ALP activity was determined using LabAssay ALP colorimetric assay kit (Wako Pure Chemicals, Osaka, Japan). The total protein content was measured at 570 nm using a commercial BCA Protein Assay kit (Beyotime, Shanghai, China) to normalize the ALP activity.

To determine the early differentiation of preosteoblasts stimulated by BioCaP alone or BioCaP with Icariin or/and BMP-2, the ALP activity and the protein content were measured after treatment on days 1, 4, and 7. ALP activity in the cell lysates was determined using the LabAssay ALP colorimetric assay kit (Wako, Osaka, Japan). The cell number was estimated by determining total protein content measured at 570 nm using a commercial bicinchoninic acid (BCA) Protein Assay kit (Beyotime, Shanghai, China). The values representing ALP activity were expressed as mmol p-NP/mg total protein.

To determine whether the early osteogenic differentiation of BMSCs was induced by BMP-2, ALP staining was performed, as previously reported (9 (link)). Following 3, 7, 14 and 21 days of culture, the cells were fixed in 10% formalin, washed with PBS and then incubated in staining solution, containing a mixture of 0.02% 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Santa Cruz Biotechnology, Inc.) and 0.03% nitro blue tetrazolium (NBT; Santa Cruz Biotechnology, Inc.) in 0.1 M TBS, which was added into 5 ml AP buffer (100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl₂ and 0.05% Tween 20, pH 9.5) and incubated for 1 h at room temperature. Furthermore, the activity of ALP and protein content of the three groups were measured on days 3, 7, 14 and 21. The cells lysates were prepared, and the activity of ALP in the lysates was determined using a Lab-Assay-ALP colorimetric assay kit (Wako Pure Chemicals, Osaka, Japan), according to the manufacturer's instructions. The total protein concentrations were determined using a commercial BCA Protein Assay kit (Beyotime Institute of Biotechnology, Shanghai, China). The activity of ALP was calculated as nmol/h phosphorylated Nitrophenol (p-NP) release and was further normalized to the cell protein content.