Phase contrast light microscopy

BMMs (1×10⁵ cells/well) were seeded in wells of 24-well plates and incubated for 24 h prior to treatment with 10 ng/ml M-CSF for 2 days followed by treatment with 30 ng/ml M-CSF + 50 ng/ml RANKL in media containing 10% FBS for 4 days. Following incubation, the cells were fixed in 3.7% formaldehyde, permeabilized with 0.1% Triton X-100 and incubated with TRAP staining solution 387-A in the dark at 37°C. Following rinsing the cells, TRAP-positive multinucleated cells were visualized by phase-contrast light microscopy (Olympus Corporation, Tokyo, Japan). Following staining, TRAP-positive multinucleated cells (3 nuclei) were counted manually in six randomly selected visual fields. TRAP activity was assessed using a TRAP staining kit and absorbance measurement at 540 nm.

A total of 3×10³ HK-2 cells were plated in 96-well plates and, after a 24 h incubation period, cells were treated with different concentrations of TGF-β1 (0, 2, 5 and 10 ng/ml) at 37°C for 72 h. Subsequently, 10 µl CCK8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to each well and incubated at 37°C for a further 2 h, and cell viability was determined by measuring the absorbance at 450 nm on a spectrophotometer. Cell morphological changes were observed by phase-contrast light microscopy (magnification ×100; Olympus Corporation).