Osteo assay surface multiple well plate

BMMs were seeded into a Corning osteo assay surface multiple well plate (Corning, NY, USA) at a density of 4×10⁴ cells/well. Either ISL (1 or 4 μg/mL), MSNs (16 or 64 μg/mL), or MSNs-ISL (16 or 64 μg/mL) was added, along with complete medium plus 30 ng/mL M-CSF and 50 ng/mL RANKL to the well. Seven days after culture, the cells were completely removed using 10% NaClO for 5 min at room temperature, and the plates were allowed to air dry at room temperature for 5 h. Bone resorption capacity was observed under an inverted microscope, and the percentage of surface area absorption on the plate was quantitatively analyzed.

These two methods have been described in detail in our previous study [15 (link)]. Briefly, matured OCs were fixed using 4% paraformaldehyde for 10 minutes, followed by permeabilization in 0.1% Triton X-100 for another 10 minutes and then incubation with phalloidin diluted solution (G1028, Servicebio, Wuhan, China) for 2 h in the dark. Nuclei were stained with DAPI staining. The F-actin belt was detected using fluorescence microscopy.
For the bone resorption assay, after BMMs fused, an equal number of fused OCs were transplanted onto Corning Osteo Assay Surface Multiple Well Plate (Corning, Inc., Corning, NY, USA). After OC adhesion, the cells were treated with 0 and 500 nM and 8 μM Vericiguat for 2-3 days. Subsequently, the cells on the plates were eliminated. The percentage of the resorbed region was randomly measured in totally three resorption sites from three independent experiments using ImageJ (National Institutes of Health, Bethesda, MD).

The bone resorption assay was described previously and conducted using the Corning osteo assay surface multiple well plate (Corning, Inc., Corning, NY, United States) (Feng et al., 2017 (link); Lee et al., 2019 (link)). Briefly, BMMs were seeded and cultured in 96-well plates (8 × 10³ per well) in α-MEM supplemented with or without CS-6 as indicated above for 10–14 days. After elimination of OCs, the plates were stained with von Kossa (GP1054, Servicebio, Wuhan, China) in order to make the bony resorption pits more clear than normal surface coating. The percentage of the resorbed areas in three random resorption sites was measured using Image J software (National Institutes of Health, Bethesda, MD).

Pit formation assay was performed using the Corning Osteo Assay Surface Multiple Well Plate (Corning, Inc., Corning, NY, USA). BMMs or RAW264.7 cells were seeded onto 96-well plates at a density of 5 × 10³ and cultured in α-MEM supplemented with stimulus as indicated in results section for 14 days. The culture medium was replaced with fresh medium containing these reagents every 2 days. After the culture, plates were stained with Von Kossa to increase contrast between pits and surface coating and observed under a light microscope. The percentage of the resorbed areas and the number of resorption pits in three random resorption sites were measured under microscopic examination using Image-Pro Plus 6.2 software (Media Cybernetics, Silver Spring, MD, USA). The assays were performed in triplicate, and a representative view from each assay is presented.

Corning Osteo Assay Surface multiple-well plates (Corning, NY, USA) were used to directly assess the osteoblastic activity in vitro as described previously [30] (link), [31] (link). In brief, the MSCs were seeded at 2×10⁴/well (1×10⁴/cm²) in a Corning Osteo Surface plate (Corning, NY, USA) with the osteogenic medium. The osteogenic medium was refreshed every 3 days. The formed bone nodules were determined on Day 18 after osteogenic differentiation commenced and were measured using von Kossa staining. Briefly, cells were washed with PBS 3 times and fixed with 4% paraformaldehyde in PBS for 45 minutes at room temperature. After washing with deionized water, the cells were stained with 5% silver nitrate (Sigma-Aldrich, MO, USA) for another 45 minutes at room temperature under a bright light. To stop the silver nitrate reaction, the cells were washed with water and treated with a 5% solution of sodium thiosulfate (Sigma-Aldrich, MO, USA). Following another water wash and air drying, the nodules were visualized as dark staining patches under a light microscope. The area of von Kossa-positive nodules was determined using the public Image J software (developed by NIH, rsb.info.nih.gov/ij/) and the total areas (µm²) of the von Kossa-positive nodules were calculated.