450 fluorescent inverted phase contrast microscope

hBMSCs were seeded into 24-well plates and cultured in OIM with PD at different doses (0, 30, 10, and 100 μM) at 37 °C and 5% CO₂ for 21 days. Then, the cells were washed with PBS twice and fixed with 97% ethanol for 10 min. The hBMSCs were then washed three times with deionized water and stained with a 0.1% Alizarin red staining solution (pH 8.3) for 30 min at room temperature. After the Alizarin red solution was removed, the hBMSCs were rinsed with deionized water and PBS twice each and then dried at room temperature. The cells were observed using a 450 fluorescent inverted phase contrast microscope (Nikon Corporation, Tokyo, Japan).

hBMSCs were seeded and cultured in the 6-well plates at a density of 1 × 10⁶ per well for osteoblastogenesis. After osteogenic induction in strategy one, alizarin red staining and ALP activity assay were performed to identify bone mineral of cells according to the manufacturers’ introduction. Specifically, cells were cultured in varying concentrations of Chrysosplenetin for 14 days and fixed with 4% PFA for 30 min, followed by PBS washing. Alizarin red staining solution was added into the wells for 5 min. The cells were viewed using a 450 fluorescent inverted phase-contrast microscope (Nikon Corporation, Tokyo, Japan).
As for ALP activity assay, hBMSCs were modulated same as what in alizarin red staining test. Then, cell lysates were collected in a test tube with alkaline solution and subjected to ALP activity analysis by a fluorometric detection kit. The absorbance set up as 450 nm was measured using a microplate reader (ELx800, BioTek, Winooski, VT, USA).