Alizarin red s

Mice were eviscerated, the skin was removed, and the resulting samples were transferred into acetone for 48 h after overnight fixation in 95% ethanol. Skeletons were then stained in Alcian blue and Alizarin Red S solution (Beyotime, Cat# C0148S, China) for 3 days at 37 °C and sequentially cleared in 1% KOH. Skeletons were then replaced in 1% KOH/20% glycerol for 3 days and passed through increasing concentrations of 1:1 glycerol/ethanol solution (20%, 50%, and 100%) for 1 day. Finally, the stained skeletons were dehydrated in glycerol for imaging and storage.

Osteogenesis was confirmed by Alizarin Red S staining. The cells were seeded on different coatings and cultured in osteogenic medium. At the indicated time point, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature and stained with 0.1% Alizarin Red S (Beyotime, China) at room temperature for 30 min. Afterward, the cells were washed twice with PBS and air dried before the ARS staining was eluted with 5% perchloric acid (SCRC, Shanghai, China). The 100 μl solution from each well was then transferred to a 96-well plate, and the optical density (OD) was measured at 490 nm using a spectrophotometer (SPECTRA MAX PLUS 384 MK3, Thermo, USA).

This study was approved by the hospital ethic committee, and the human-related experiments were performed following the Declaration of Helsinki protocol. The primary CD 138⁺ BM cells were isolated from mononuclear cells of the patients and normal donors. The BM-MSCs obtained from the patients and health donors were cultured with Dulbecco's modified Eagle medium (DMEM) with 20% fetal bovine serum (FBS).
After cell attachment, BM-MSCs were cultured with the osteogenic induction medium (full culture medium containing 10⁻² M β-sodium glycerophosphate, 50 mg/mL L-ascorbic acid, and 10⁻⁷ M dexamethasone) to induce the progression of osteoblast differentiation. After culture for 21 days, BM-MSCs were strained with alizarin red S (Beyotime), and then, the osteoblast mineralization in three random microscopic regions was imaged and quantified with Image-Pro Plus 6.0 software. Finally, the relative mineralized regions were calculated.

HEK-293, C3H10T1/2, and C2C12 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The above cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), which contained 10% (v/v) fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL). Cell culturing dishes, flasks, 6-well plates, and 24-well plates were incubated at 37 °C with 5% CO₂. DMEM, FBS, and trypsin were purchased from Gibco (Chinese branch). BMP9 (sc-514211), ALP (sc-271431), RUNX2 (sc-12488), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-32233) antibodies, retinoic acid receptor beta (RARβ) specific inhibitor Le135 (sc-204053), and ATRA (sc-200898) were bought from Santa Cruz Biotechnology (Chinese branch). Osteopontin (OPN, ab8448) and RARβ (ab53161) antibodies were purchased from Abcam (Chinese branch). Serpina3n (AF4709) antibody was sourced from R&D systems (Chinese branch). Dexamethasone (D4902) was obtained from Sigma-Aldrich (Chinese branch). Dylight 594 conjugated secondary antibody (A23410) was purchased from Abbkine (Chinese branch). Alkaline Phosphatase Assay Kit (C3206) and Alizarin Red S (ST1078) were bought from Beyotime (Shanghai, China).

Extracellular matrix mineralization was assayed with an alizarin red S (C0148S, Beyotime Biotechnology, China). Seed 5 × 10⁴ cells/well in 6-well culture dishes. After cell apposition and dissemination for 24 h, H₂O₂ treatment was performed by 200 μm H₂O₂ treatment for 4 h; then, PBM irradiation was performed for 630 s. After 48 h, cells were cultured for 21 d using osteogenic induction medium; and the medium was changed every 3 d. The cells were rinsed in PBS without calcium and magnesium after medium removal. The cells were fixed in fixative for 20 min and washed in PBS three times. The cells were stained with alizarin red Stain solution for 30 min. The last step was to wash the cells using deionized water; next, all cells were photographed with microscope.

MC 3T3 E1 cells or BMSCs were fixed in ethanol for 30 min at 4 °C after induced by osteogenic medium for 21 days. Alizarin Red S solution (Beyotime, Shanghai, China, C0148S) was added to the cells for 30 min at 37 °C, and the cells were flushed with distilled water at least 3 times before being photographed. A 5% perchloric acid solution was added into each well to dissolve calcium nodules at 37 °C for 30 min to quantify calcium nodules, the absorbance was measured at 490 nm. The relative expression result is represented by the fold change.