Alizarin red staining kit

2 × 10⁴/ml MC3T3-E1 cell was inoculated in transwell lower chamber in a 6-well plate, different DDM-FG compounds (DDM-FG1, 1 g DDM with 0.1 ml FG, DDM-FG2, 1 g DDM with 0.5 ml FG) were prepared in transwell upper chamber and cultured in an incubator at 37 °C (As shown in Fig. 6E). At 14 and 21 days, the different materials in the upper chamber were discarded, the cells in the lower chamber were dyed with 0.1% alizarin red for 5 min follow the Alizarin Red staining kit (Beyotime Biotechnology, China) instruction. After taking photograph of the nodules, 10% acetylpyridine was added to the 6-well plate, 500 μl/well, and incubate the solution at 37 °C for 15 min to dissolve mineralized nodules. Lastly, the incubated solutions were measured at 570 nm of absorbance with the microplate reader (Muliskan, Thermo, USA). The test was independently repeated three times.

We allowed Morusin, purchased from MedChemExpress (MCE, New Jersey, USA), to dissolve in dimethylsulfoxide (DMSO) and dilute in cell culture solution to make the DMSO content less than 0.1% of the total amount. We procured the penicillin/streptomycin, Trizol, and reverse transcription kit from Thermo Fisher Scientific (Waltham, USA). We ordered the α-Minimum Essential Medium (α-MEM) and Fetal Bovine Serum (FBS) from HyClone Co. (Logan, USA). Rat Dickkopf-related protein 1 (DKK1) was obtained from R&D Systems (Waltham, USA) [18 (link)]. The antibody for runt-related transcription factor 2 (RUNX2, sc-390351) was ordered from Santa Cruz Biotechnology Co. (Dallas, USA). The collagen type I alpha 1(COL1A1, A16891), phosphorylated β-catenin (p-β-catenin, AP0579), and β-catenin (A19657) antibody was obtained from ABclonal Technology Co., Ltd. (Wuhan, China). The alizarin red staining kit and alkaline phosphatase chromogenic kit was ordered from Beyotime Co., Ltd. (Shanghai, China). The synthesis of all primers was performed by Tianyihuiyuan Biotech Co., Ltd. (Wuhan, China). As to other reagents, we selected the ones of analytical grade for use in experiments.

Third-generation BMSCs in an appropriate growth state were adjusted to 2 × 10⁴ cells/well (6-well plate) with complete medium, and the culture medium was replaced with osteogenic induction medium (50 μg/mL vitamin C; Sigma-Aldrich, St. Louis, MO, USA), 5 mM β-sodium glycerophosphorin (Sigma-Aldrich), and 0.1 μmol/L dexamethasone (Sigma-Aldrich). Therefore, vitamin C needed to be administered immediately. Half the volume of the liquid was changed every alternate day. Approximately 14 days after induction, the cell morphology was observed under a microscope, and ALP staining was performed using an ALP staining kit (Beyotime Biotechnology, Shanghai, China). The induction was continued for 21 days, and alizarin red staining was performed using an alizarin red staining kit (Beyotime Biotechnology, Shanghai, China).

ALP staining was carried out after six days of culture. Cells were stained with the ALP color development kit (Beyotime, Shanghai, China) according to the manufacturer’s protocols after being fixed with 4% paraformaldehyde for 10 min. After being stained for 15 min, cells were washed with PBS three times. Subsequent observation and image capture was carried out under phase-contrast microscopy. For Alizarin red staining, cells were harvested after 14 days, fixed in 4% paraformaldehyde for 10 min and stained with Alizarin red staining kits (Beyotime, Shanghai, China) for 60 min. Subsequent observation and image capture were carried out under phase-contrast microscopy.

BMSCs at a density of 2 × 10⁴ cells/well were seeded in 24-well plates. After 24 h, the baseline medium was replaced with osteogenic induction medium containing various concentrations of leonurine (0, 2, 5, and 10 μM). The cells were cultured in osteogenic induction medium for either 6 days (ALP staining) or 14 days (Alizarin red staining).
ALP staining was conducted to ascertain the effect of leonurine on BMSC differentiation. In brief, BMSCs were harvested after 6 days of culture and fixed with 4% paraformaldehyde for 10 min. An ALP color development kit (Beyotime, Shanghai, China) was used in the study according to the manufacturer’s protocols. Briefly, the cells were stained for 15 min and washed three times with PBS. The stained cells were subsequently observed under phase-contrast microscopy, with representative images captured.
Alizarin red staining was further performed to determine the degree of calcium deposition in BMSCs between the leonurine treatment groups at various concentrations. Briefly, BMSCs were harvested after 14 days of culture in osteogenic medium as outlined above. After fixation in 4% paraformaldehyde for 10 min, the cells were stained with Alizarin red staining kits (Beyotime, Shanghai) for 60 min and washed three times with ddH₂O.
The stained cells were subsequently observed under phase-contrast microscopy, with representative images captured.