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J Cell Physiol. 2018 Dec 4. doi: 10.1002/jcp.27852. [Epub ahead of print]

Evaluation of efficacy on RANKL induced osteoclast from RAW264.7 cells.

Author information

1
Department of Spine surgery, Honghui Hospital, School of Medicine, Xi'an Jiaotong University, Shaanxi, China.
2
Department of Orthopedics, The Second Affiliated Hospital of Harbin Medical University, Harbin, China.
3
The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Heilongjiang, China.
4
Department of Neuroscience, Johns Hopkins University, Baltimore, Maryland.

Abstract

Established RAW264.7 cell lines for osteoclastic differentiation has been widely engaged in bone homeostasis research, however, the efficacy of RANKL independently stimulating has rarely been defined, because protocols were usually developed and modified by various laboratories. Otherwise, problematic issues are also lie in the cell's seeding density, RANKL stimulating time point, and distinguishing osteoclastogenesis ability of RANKL-treated RAW264.7 cells. Therefore, in the current study, we examined the efficacy of various concentrations of RANKL-treated RAW264.7 for its osteoclastic differentiation with or without pretreated other costimulators such as: LPS and/or M-CSF. The oteoclastogenesis ability of RANKL-treated RAW264.7 cells was demonstrated by bone resorption pit, F-actin, and osteoclastogenesis specific marker studies. Besides that, through tartrate-resistant acid phosphatase (TRAP) staining, we clarified to start the treatment with 30 ng/ml RANKL at 12 hr after seeded RAW264.7 with the density of 6.25 × 10 3  cells/cm 2 manifested an significantly increased number of multinucleated osteoclastic cells. Overall, our results establishing an optimal method for RANKL independently inducing RAW 264.7 cell osteoclastic differentiation, which could efficiently generate osteoclasts in vitro for significant advances in our understanding of bone biology.

KEYWORDS:

RANKL; RAW264.7; bone marrow macrophages; osteoclast; osteoclastogenesis; protocol

PMID:
30515780
DOI:
10.1002/jcp.27852

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