Recombinant murine rankl

Murine BMMs at a density of 1 × 10⁶ cells/ml or RAW264.7 cells at a density of 1 × 10⁵ cells/ml were plated in a chamber slide (Thermofisher, Waltham, MA, USA) and cultured in complete α-MEM supplemented with 50 ng/ml murine recombinant M-CSF, 25 ng/ml murine recombinant RANKL (Peprotech, Rocky Hill, NJ, USA) in the presence or absence of 20 μg/ml exosomes, a concentration used previously in multiple studies^{27 (link),66 (link),81 (link),82 (link)}. On day 4, TRAP staining was performed using a leukocyte acid phosphatase kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. The multinucleated TRAP-positive cells from five biological replicates were considered mature OC cells and monitored by Zeiss Axioplan2 microscope. The size of OC cells was measured by ImageJ as reported^{83 (link)}.

The RAW264.7 murine monocytic cell line was purchased from KeyGEN Biotech Co., Ltd. (Nanjing, China). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 5.6 mM D-glucose (Hyclone, Logan, UT, USA) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum (FBS; Hyclone), and incubated at 37°C in 5% CO₂ humidified air. To investigate the effect of high glucose on osteoclastogenesis, the cells were seeded at a density of 1×10⁵ cells/well in 6-well plates, 2×10⁴ cells/well in 24-well plates and 5×10³ cells/well in 96-well plates, and cultured in DMEM with different doses (5.6 and 20.2 mM) of D-glucose or DMEM with mannitol (14.6 mM; Bio Science Technology. Co., Ltd., Shanghai, China) added at 37°C in 5% CO₂ humidified air for 4 or 5 days. Following attachment of the cells, 100 ng/ml murine recombinant RANKL (PeproTech, Rocky Hill, NJ, USA) was added to the different mediums to generate osteoclasts. The cells cultured with 5.6 mM glucose in the absence of RANKL were defined as the untreated group, those cultured with 5.6 mM glucose in the presence of RANKL were defined as the control group, those cultured with 20.2 mM glucose were defined as the high glucose group and those cultured with mannitol were defined as the osmotic control group.

To determine the role of exosomal miR-19a in the bone resorption activity of OC cells, pit assay was performed on bone slices (Immunodiagnostic Systems). 50,000 bone marrow-derived OC precursor BMMs were seeded on sterilized bone slices placed in a 96-well plate. The BMMs were cultured with complete α-MEM supplemented with 50 ng/ml murine recombinant M-CSF, 25 ng/ml murine recombinant RANKL (Peprotech, Rocky Hill, NJ, USA) in the presence or absence of 20 μg/ml exosomes. The media was changed every 2–3 days. After two weeks of cell culture, cells were washed, and resorption pits were stained with 1% toluidine blue. The resorption area was photographed under microscopy and the image was analyzed by ImageJ.