Alizarin red staining kit

Traditional Chinese medicine ZG was obtained from Sanjiu Medical & Pharmaceutical Co., Ltd. (Beijing, China). It was dissolved in normal saline as a vehicle for animal experiment while using phosphate-buffered saline (PBS) as vehicle for cell experiment. Fetal bovine serum (FBS) was purchased from Gibco (United States). Alpha-minimal essential medium (a-MEM), PBS, trypsin−EDTA, penicillin, and streptomycin were purchased from Wisent (Canada). Cell Counting Kit-8 (CCK-8) and MHY1485 were from MCE (China). IGF-1, M-CSF, RANKL were from R&D systems (United States). Osteogenic Differentiation Medium and Alizarin Red staining kit were purchased from Cyagen (China).

hSCAPs were cultured in osteogenic media for 4 or 14 days and fixed with 4% paraformaldehyde.^{45 (link)} The ALP staining kit (Byotime Biotech, Shanghai, China) and alizarin red staining kit (Cyagen, CA, USA) were used in line with the manufacturers’ instructions.

Oil red O staining was performed according to the manufacturer’s protocol for the oil red O staining kit (Cyagen; Suzhou; China). Cells cultured in a 6-well-plate were rinsed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (Tiengen Biological, China) for 10 min, and washed with 60% isopropyl alcohol (Tiengen Biological). Cells were then incubated in 2% (w/v) oil red O staining solution (Cyagen) for 1 h at room temperature. Excess staining was removed by washing with 60% isopropyl alcohol, followed by several washes with distilled water. Lastly, images were collected with an Olympus microscope (BX51, Tokyo; Japan).
Alizarin red staining was performed according to the manufacturer’s protocol for the alizarin red staining kit (Cyagen). Cells cultured in a 6-well-plate were rinsed three times with PBS, fixed with 4% formaldehyde for 10 min at room temperature, and incubated with fresh alizarin red staining solution for 5 min, and then, images were collected with an Olympus microscope (BX51).