Three- to 4-week-old female C57BL/6 J mice were euthanized by carbon dioxide and bilateral thoracotomy.

Isolation of osteoprogenitor cells and culture. Osteoprogenitor cells were isolated as previously described with modifications. All buffers were ice cold, unless otherwise indicated. Briefly, the femur, tibia, humerus, radius, and ulna of mice were harvested. BM was flushed out and discarded. The bone was collected and cut into 1-mm3 chips in harvest buffer and then digested in digestion buffer containing growth media [α minimum essential medium (αMEM), fetal bovine serum (FBS) (10%, v/v), penicillin/streptomycin (10,000 U/ml; 1%, v/v)], and collagenase type 2 (1 mg/ml, w/v; Worthington Biochemical, Lakewood, NJ) while shaking at 60 rpm on orbital shaker (catalog no. 51700-13, Cole-Parmer, Vernon Hills, IL) in humidified incubator (37°C, 5% CO2) for 1.5 hours. Digested bone chips were rinsed three times with growth media and transferred to two wells of six-well plate for culture. Media were replaced after 3 days, and bone chips were further cultured for 3 days for cells to adhere and proliferate before passage. For the first passage, cells were treated with 0.25% trypsin-EDTA (Thermo Fisher Scientific) for 3 min, neutralized with growth media, detached with cell scraper, and subcultured along with bone chips. The procedure for subsequent passages were similar but without bone chips. All experiments were performed at three to four passages. For estradiol (E2) withdrawal experiments, cells were cultured in the absence or presence of E2 (Sigma-Aldrich) supplemented at 100 nM every 3 hours to charcoal-stripped growth media [αMEM, charcoal-stripped FBS (10%, v/v), penicillin/streptomycin (10,000 U/ml; 1%, v/v)]. For adenosine supplementation, cells were supplemented with adenosine (30 μg/ml; Sigma-Aldrich) in growth media with fresh changes of media every day.

Isolation of mononuclear cells and macrophage/osteoclast differentiation. All buffers used were ice cold, unless otherwise indicated. Briefly, the femur, tibia, humerus, radius, ulna, and vertebra were harvested and crushed with pestle and mortar in harvest buffer [phosphate-buffered solution (PBS) and FBS (2%, v/v)] to release BM tissue. BM was passed through a 70-μm cell strainer and centrifuged at 200g for 5 min. Cells were resuspended in harvest buffer, gently layered onto Ficoll-Paque PLUS (GE Healthcare, Marlborough, MA) at 1:1 ratio, and centrifuged without rotor acceleration and deceleration at 200g for 15 min. Afterward, the opaque middle layer with cells was collected, washed with harvest buffer, and centrifuged at 200g for 5 min to yield a cell pellet. Isolated mononuclear cells were cultured in macrophage induction media, containing growth media, prostaglandin E2 (PGE2; 10−7 M; Santa Cruz Biotechnology, Dallas, TX), and M-CSF (10 ng/ml; PeproTech, Rocky Hill, NJ) at 100,000 cells/cm2. For osteoclast differentiation, macrophages cultured for 3 days were further induced in osteoclast induction media containing growth media, PGE2 (10−7 M), M-CSF (10 ng/ml), and RANKL (10 ng/ml; PeproTech). For estradiol (E2; Sigma-Aldrich) withdrawal experiments, osteoclasts were cultured in the absence or presence of E2 supplemented at 100 nM to media containing charcoal-stripped growth media, PGE2 (10−7 M), M-CSF (10 ng/ml), and RANKL (10 ng/ml; PeproTech). For adenosine supplementation, cells were supplemented with adenosine (30 μg/ml; Sigma-Aldrich) in differentiation media with fresh media change every day.

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