Acid phosphatase staining kit

The TRAP assay was performed using an acid phosphatase staining kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. For osteoclastogenesis experiments, the indicated number of RAW264.7 mouse macrophage-like cells was seeded on a 12-well tissue culture plate with MEMalpha media supplemented with 10% FBS, 100 IU·mL⁻¹ penicillin and 100 μg·mL⁻¹ streptomycin. Raw cells were then treated with 20 ng·mL⁻¹ mouse RANKL (R&D Systems, Inc., Minneapolis, MN) with or without conditioned media derived from MC3T3 cells. After 4 days of cell culture and osteoclast generation, the media were removed and washed three times with PBS. Cells were fixed with a fixing solution supplied by the manufacturer. The cells were incubated at 37 °C with a solution containing deionized water, Fast Garnet GBC, napthol phosphate, acetate, and tartrate for 1 h. The staining solution was removed, washed with PBS, and air-dried. TRAP-positive cells were collected using a Cytation 5 Imaging Reader (Biotek, Winooski, VT). TRAP-positive cells with three or more nuclei across the culture area were counted as multinucleated osteoclasts.

Bone marrow macrophage from tibia and femur of WT and cKO mice were prepared as previously described (He et al., 2019 (link)). BMMs were then seeded on to 96-well plate (6 × 10³ cells/well) and cultured in α-MEM supplemented with 30 ng/mL M-CSF (416-ML, R&D system, Minneapolis, MN, United States), and 100 ng/mL RANKL (462-TEC, R&D system). The media was replaced every 2 days and after 7 days of culture the cells were fixed and stained with Acid Phosphatase Staining kit (387A, Sigma-Aldrich, St. Louis, MO, United States) according to the protocol of the manufacturer. TRAP positive multinucleated cells with more than three nuclei were counted as OCs.

Murine leukemia monocyte RAW 264.7 cells (ATCC, TIB-71, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, ATCC), supplemented with 10% FBS (Gibco, Carlsbad, CA, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, Carlsbad, CA, USA). Osteoclast differentiation medium, composed of phenol-red free MEMα with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 100 ng/mL receptor activator of NF-κB ligand (RANKL, Millipore GF091, Billerica, MA, USA), were used to induce the differentiation of RAW 264.7 cells during EAF treatment. For cell proliferation studies, RAW 264.7 cells were treated with phenol-red free MEMα medium with 10⁻⁸ M E2, vehicle, and 10⁻¹⁰ to 10⁻⁶ M of EAF for 24 h. The cell viability was determined by MTS assay as described above. To determine the effects on differentiation, RAW 264.7 cells were induced by differentiation medium containing RANKL and treated with 10⁻⁸ M of E2, 10⁻¹⁰ to 10⁻⁶ M of EAF, or its vehicle for five days. Treated cells were stained for Tartrate Resistant Acid Phosphatase (TRAP) expression by using an acid phosphatase staining kit (Sigma). TRAP-positive multinucleated cells showing more than three nuclei were counted as mature osteoclasts. The TRAP activities of RAW 264.7-derived osteoclasts were measured by the acid phosphatase assay kit (BioVision, Milpitas, CA, USA).