Alizarin red s staining kit

Ginsenoside Rg1 (purity ≥ 98%, molecular structural formula is shown in Supplementary Figure S1) was obtained from Shanghai Tubei Biological Co., Ltd. The MTT kit was obtained from Sigma (United States). Annexin V-FITC/PI kit was obtained from BestBio (Shanghai, China). PI kit and RIPA lysis buffer were obtained from Beyotime Biotechnology (Shanghai, China). The BCIP/NBT alkaline phosphatase chromogenic kit and Alizarin Red S staining kit were obtained from Solarbio (Beijing, China). SYBR Green I Master Mix and reverse transcription kit were obtained from Thermo (United States). Anti-VEGF antibody was obtained from Boster (Wuhan, China). Anti-NOG, anti-Notch1, anti-CD31 and anti-Osterix antibodies were obtained from Abcam (United Kingdom). Anti-Emcn antibody was obtained from Abbkine (Wuhan, China). Horseradish enzyme-labeled goat anti-rabbit IgG and horseradish enzyme-labeled goat anti-mouse IgG were obtained from ZSGB-Bio (Beijing, China). Anti-GAPDH antibody, fluorescein (FITC)-conjugated AffiniPure goat anti-rabbit IgG and Cy3-conjugated AffiniPure goat anti-mouse IgG were obtained from Proteintech (United States). Rabbit anti-CD73/PE conjugated antibody, rabbit anti-CD90/PE conjugated antibody, rabbit anti-CD14/FITC conjugated antibody and rabbit anti-CD19/FITC conjugated antibody were obtained from Bioss (Beijing, China).

The hBMSCs were inoculated in 24-well plates. When cell confluence reached 80%, the medium was changed to osteogenic differentiation medium (Cyagen, USA). The medium was changed every 3 days and when the medium was changed, exosomes were added to the medium. Alkaline phosphatase (ALP) staining and Alizarin Red S staining were done to evaluate the level of osteogenic differentiation. After the hBMSCs were cultured in osteogenic differentiation medium for 7 days, ALP staining was conducted according to the manufacturer’s instructions (Beyotime, China). The cells were incubated with 10 mM p-nitrophenyl phosphate (Meilunbio, China) as the substrate at 37 °C for 15 min. Afterward, ALP activity was quantified at 420 nm by a microplate reader [34 (link)]. After cultured in osteogenic differentiation medium for 3 weeks, the hBMSCs were fixed, stained, and cleared according to the instructions of the Alizarin Red S staining kit (Solarbio, China). Based on the previously reported methods, the stained mineralization nodules were dissolved with 10% cetylpyridinium chloride at 37 °C for 30 min [35 (link)]. The solution was added to a 96-well plate, and a microplate reader was used detect the OD value at 562 nm for quantitative analysis.