Trap staining kit

CD14⁺ monocytes were prepared from PBMCs using microbeads (Miltenyi Biotec, Auburn, CA, USA). Human CD14⁺ monocytes were seeded in 48-well plates at 5 × 10⁴ cells/well in 1 mL of medium. Monocytes were cultured in α-minimum essential medium supplemented with 10% heat-inactivated foetal bovine serum and 25 ng/mL recombinant human macrophage colony-stimulating factor (rhM-CSF, R&D system) for 3 days. Monocytes were then cultured under RANKL (30 ng/mL) + M-CSF (25 ng/mL) or IL-17A (50 ng/mL) + M-CSF (25 ng/mL) for 10 to 14 days. IL-18BP (0, 10, 50, or 100 ng/mL) was also added three days after rhM-CSF stimulation to evaluate its effect on osteoclastogenesis. After 10–14 days of RANKL + M-CSF + IL-18BP or IL-17A + M-CSF + IL-18BP stimulation, tartrate-resistant acid phosphatase (TRAP)-positive cells were identified using a TRAP staining kit (Cosmo Bio, Tokyo, Japan), as described previously [20 (link), 21 (link)].

CD14⁺ monocytes were prepared from the PBMCs of healthy donors (n = 6) using microbeads (Miltenyi Biotec, Auburn, CA, USA). Human CD14⁺ monocytes were seeded in 24-well plates at a density of 1 × 10⁶ cells/well in 1 mL of α-minimum essential medium supplemented with 10% heat-inactivated fetal bovine serum and 25 ng/mL recombinant human macrophage colony-stimulating factor (rhM-CSF, R&D system) for 3 days. Monocytes were cultured with RANKL (20 ng/mL), M-CSF (25 ng/mL), IL-17A (50 ng/mL), and M-CSF (25 ng/mL) for 10–14 days. DJ-1 was added at 0, 10, 50, and 100 ng/mL to evaluate its effect on osteoclastogenesis. The culture medium was changed every 3 days. After 10–14 days, tartrate-resistant acid phosphatase (TRAP)⁺ cells were identified using the TRAP staining kit (Cosmo Bio, Tokyo, Japan), as previously described^{37 (link)}. A bone resorption assay kit (Cosmo Bio) was used to evaluate the effects of DJ-1 on the functional abilities of osteoclasts. CD14⁺ monocytes (n = 5) were plated on a bone plate under M-CSF (25 ng/mL) and RANKL (20 ng/mL) stimulation with various concentrations of DJ-1 (0, 10, 50, and 100 ng/mL) and then cultured for 10–14 days. The resorption pit area (%) was calculated by using ImageJ software.

Thiazolyl blue tetrazolium bromide and risedronate were purchased from Sigma‐Aldrich Inc. The TRAP staining kit was purchased from Cosmo Bio Co. Dulbecco's Modified Eagle Medium (DMEM), hematoxylin and eosin (H&E), 4′,6‐Diamidino‐2‐Phenylindole (DAPI), as well as p38 MAPK beta and AP1/JUN/RUNX2/OCN antibodies were purchased from Thermo Fisher Scientific Inc. Integrated DNA Technologies provided the primers (NFATc1, TRAP, GAPDH (Mouse), ALP, OPG, and GAPDH (Human)). AMRESCO provided a phenol‐free total RNA purification kit.

All fixed rat calvaria samples were decalcified with RapidCal immune (BBC Biochemical) solution with gentle shaking at 4 °C. The solution was changed every 2 days. After 21 days, the samples were dehydrated and embedded into paraffin. Next, 10 µm thick coronal sections were executed. The sectioned slides were stained with Hematoxylin & Eosin (Abcam, UK), Masson's Trichrome staining kit (VitroVivo Biotech), and immunohistochemistry staining (OCN, sc‐365797, Santacruz, TX, USA, 1:100). TRAP staining was performed with paraffin slides using TRAP staining kit (PMC‐AK04‐COS, Cosmo bio, Japan) following the provided protocol.

Osteoclastogenesis was evaluated by quantifying cells positively stained for TRAP. Briefly, the cells on 96-well plates were fixed with a 10% neutral formalin buffer solution for 5 min and then stained using a TRAP staining kit (Cosmo Bio, Tokyo, Japan). Under a light microscope, osteoclasts were observed as TRAP-positive with three or more multinuclear cells, and their total number was counted in each well.

THP-1 human monocytes were purchased from ATCC (Manassas, VA, USA). Biological passage number 8 was used to culture the THP-1 cells in RPMI1640 (Welgene, Gyeongsan, Korea) containing 10% FBS, 2 mM L-glutamine, and 1% penicillin–streptomycin. Cells were seeded in 6-well plates (Nunc, Rochester, NY, USA) at the density of 3 × 10⁵ cells/well and differentiated into M0 macrophages using phorbol 12-myristate 13-acetate (Sigma-Aldrich Chemical, St. Louis, MO, USA) for 48 h. Then, the M0 macrophages were differentiated into osteoclasts in high-glucose DMEM (Gibco, Amarillo, TX, USA) containing 1% penicillin-streptomycin (Gibco, Amarillo, TX, USA), 10% fetal bovine serum (Alphabioregen, Boston, MA, USA), 50 ng/mL RANKL (R&D Systems, Minneapolis, MN, USA), and 50 ng/mL M-CSF (R&D Systems, Minneapolis, MN, USA) for 14 days. The differentiation medium was replaced with fresh medium every 2 days. TRAP-positive osteoclasts were validated using a TRAP staining kit (Cosmo Bio, Tokyo, Japan) according to the manufacturer’s protocol.

Cells were fixed and stained with a TRAP staining kit (Cosmo Bio) for 60 min at 37 °C. Staining was quantified using ImageJ software (National Institutes of Health).