Isolation and Characterization of Osteoclasts

Fresh bone marrow macrophages (BMMs) were isolated from C57BL/6 mice using the methods approved by University of Western Australia Animal Ethics Committee (RA/3/100/1244) as described 33 (link), 34 (link), and grown in culture medium (25 ng/ml of M-CSF, 100U/ml of Penicillin/Streptomycin and 10% FBS in α-MEM) in T75 flasks. After the cells were confluent they were removed from the flask using Tryple reagent (Thermofisher, Scoresby, Australia) and scraping, and then seeded into a 96-well plate at 6×10³ cells per well with culture medium overnight. The next day, BMMs were stimulated with RANKL at the concentration of 50 ng/ml and the presence of LrB or other compounds, and then medium replaced every two days until osteoclasts formed. After 5 days the cells were then fixed with 2.5% glutaraldehyde in phosphate-buffered saline (PBS) for 10 minutes and stained for tartrate-resistant acidic phosphatase (TRAcP) activity. TRAcP positive multinucleated cells (MNCs) were scored as osteoclast-like (OCL) cells if they had three or more nuclei.

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Liu Y., Wang C., Wang G., Sun Y., Deng Z., Chen L., Chen K., Tickner J., Kenny J., Song D., Zhang Q., Wang H., Chen Z., Zhou C., He W, & Xu J. (2019). Loureirin B suppresses RANKL-induced osteoclastogenesis and ovariectomized osteoporosis via attenuating NFATc1 and ROS activities. Theranostics, 9(16), 4648-4662.

Publication 2019

Tryple reagent

Animal ethics committee Bone marrow C57bl mice Cells Glutaraldehyde M csf Macrophages Nuclei Osteoclast Penicillin Phosphate Rankl Saline Streptomycin Tartrate resistant acidic phosphatase

Corresponding Organization :

Other organizations : First Affiliated Hospital of Guangzhou University of Chinese Medicine, University of Western Australia, Guangzhou University of Chinese Medicine, Guangxi Medical University

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Variable analysis

independent variables

RANKL concentration (50 ng/ml)
Presence of LrB or other compounds

dependent variables

Osteoclast-like (OCL) cell formation (TRAcP positive multinucleated cells with three or more nuclei)

control variables

Bone marrow macrophages (BMMs) isolated from C57BL/6 mice
Culture medium (25 ng/ml of M-CSF, 100U/ml of Penicillin/Streptomycin and 10% FBS in α-MEM)
Cell seeding density (6×10^3 cells per well)
Cell culture duration (5 days)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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