Formaldehyde

Alizarin red S (Sigma-Aldrich) was solubilized with distilled water. For mineralization analysis, C3H10T1/2 cells were treated with 50 μM KP-10 or transfected with pCMV-NFATc4 (0.4 g) for 20 days and then fixed with 4% formaldehyde (Duksan Pure chemicals Inc.) for 5 min. After washing with distilled water, cells were stained with 300 μg/mL of Alizarin red S solution for 30 min at room temperature. And washing with distilled water. Staining was then documented with an Epson perfection V37 scanner (Seiko Epson, Suwa, Japan).

The activity of ALP was assessed as an early marker of osteogenic differentiation at day 6 after differentiation induction. Cells plated into 96-well plates were washed twice with PBS (phosphate buffered saline) and then fixed with 4% formaldehyde (Duksan, Gyeonggi-do, Korea) for 30 min at room temperature. ALP activity was determined colorimetrically by incubating cells with the substrate p-nitrophenyl phosphate (Sigma-Aldrich) diluted in a 1:15 ratio in alkaline buffer solution 1.5 M, pH 10.3 (Sigma-Aldrich) at 37 °C for 30 min. The absorbance was measured at 405 nm and normalized against each experimental group's MTT value with a Multiskan^TM GO microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Values were expressed as the fold change relative to control (undifferentiated) cells.

Bone marrow macrophage cells (BMMs) were isolated from femurs and tibias of 8- to 10-week-old C57BL/6J females, as described previously [45 (link)]. Briefly, the cells were cultured in alpha minimal essential medium (a-MEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS containing 30 ng/mL M-CSF (PeproTech, NJ, USA) for three days. To differentiate into osteoclasts, BMMs were incubated in 30 ng/mL M-CSF and 50 ng/mL RANKL (PeproTech, NJ, USA) containing medium for three to four days. The differentiated cells were fixed on day 3 or day 4 with 4% formaldehyde (Duksan, Korea) with 10 min incubation at room temperature. The fixed cells were stained for tartrate-resistant acid phosphatase (TRAP) activity (Cosmo Bio Co., Ltd., Tokyo, Japan). TRAP-positive osteoclast cells with more than three nuclei were counted, and the area of TRAP-positive cells was measured using Image J software.