Fluoview 500 microscope

TRAP staining was performed as described previously [12 (link)]. Briefly, RAW264.7 cells cultured in 96-well plate was fixed with 4% Paraformaldehyde at room temperature for 20 min. After washing, the cells were stained with TRAP staining solution at 37 °C for about 20 min with close observation. Then, the TRAP staining solution was removed and the cells were washed twice with PBS. Lastly, the PBS containing sodium azide was used to preserve the samples. Samples were observed by an Olympus Fluoview 500 microscope (Japan).

RAW264.7 or BMM cells cultured in 96-well plate were washed with phosphate buffered saline (PBS), fixed in 4% Paraformaldehyde for 20 minutes at room temperature, and washed thrice with PBS. 100 μl TRAP staining solution was added for incubation at 37 °C until cells were correctly stained (approximately 20 minutes after). TRAP staining solution was removed, and cells were washed trice with PBS, Stored plates at 4 °C with PBS in wells. Samples were examined under an Olympus Fluoview 500 microscope.

Tet-PCNA KD cells (RAW264.7 cells with a Tet-on inducible PCNA knockdown) were seeded at 5000 cells/well on the Corning Osteo Assay Surface multiple well plates (Corning® Osteo Assay Surface, Corning, USA). 12 h later, the cells were simultaneously treated with Tet (20 μg/mL), RANKL (100 ng/mL) and M-CSF (50 ng/mL) for 10 days, changing the medium every three days, to induce the knockdown of PCNA, differentiation of osteoclasts and osteoclast-mediated bone resorption on the synthetic surface of Corning multiple well plates. Tet-PCNA KD cells treated only with RANKL and M-CSF but not with Tet were set as control. Then, 100 μL of 10% bleach solution was added to the surface of each well for 5 min at room temperature according to the manufacturer’s instructions. Individual resorption pits or multiple pit clusters were observed using an Olympus Fluoview 500 microscope (Japan) at 25x magnification. ImageJ software was used to analyze the area of total pits.