Osteoblast mineralized nodule staining kit

After transfection with miR-137 mimics or inhibitors, early mineralization was observed by the BCIP/NBT ALP staining kit (Beyotime) following the manufacturer's instructions. The cells were fixed with 4% paraformaldehyde (Beyotime) for 10 min, washed three times with PBS, and then incubated in BCIP/NBT staining solution at 37 °C in the dark for 30 min. After two washes with distilled water, the cells were observed and photographed, and the ALP concentration was quantified. Late-stage mineralization was determined under the instructions of the osteoblast-mineralized nodule staining kit (Beyotime). After fixation with 4% paraformaldehyde for 10 min, the cells were washed three times with PBS and stained with Alizarin Red for 10 min. Next, the cells were washed again with distilled water, followed by microscopy and photography. The calcium nodule level was quantitatively analyzed at last.

Differentiation of hBMSCs was evaluated using alkaline phosphatase (ALP) staining, ALP activity detection, and alizarin red staining (ARS). ALP staining was performed as previously described (Wu et al., 2012 (link)) using an ALP staining kit (Beyotime, Shanghai, China). ALP activity was measured using an ALP Activity Assay Kit (Elabscience, Wuhan, China). ARS was performed using an osteoblast mineralized nodule staining kit (Beyotime, Shanghai, China). The detailed steps are provided in the Supplementary Material.

The early mineralization was determined based on the instructions of BCIP/NBT alkaline phosphatase chromogenic kit (Beyotime, Shanghai, China). Cells were fixed in 4% paraformaldehyde (Beyotime) for 10 min, washed three times with PBS, and then incubated with BCIP/NBT staining solution for 30 min at 37 °C in the dark. After that, the treated cells were washed twice with distilled water and photographed. Then, ALP concentration was quantitatively analyzed. Late mineralization was measured according to the instructions of the Osteoblast Mineralized Nodule Staining Kit (Beyotime). The cells were fixed with 4% paraformaldehyde for 10 min, washed with PBS for another three times, and stained with alizarin red for 10 min. Subsequently, the treated cells were washed with distilled water to be observed and photographed microscopically, and the calcium nodules level was then quantitatively analyzed.