PMID- 36395187
OWN - NLM
STAT- MEDLINE
DCOM- 20221121
LR  - 20230105
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 17
IP  - 11
DP  - 2022
TI  - Evaluation of culture conditions for osteoclastogenesis in RAW264.7 cells.
PG  - e0277871
LID - 10.1371/journal.pone.0277871 [doi]
LID - e0277871
AB  - Osteoclasts are the only multinucleated cells in vivo responsible for bone 
      resorption and are vital for regulating bone remodeling and maintaining bone 
      mass. The RAW264.7 cell line is widely used to study osteoclastic differentiation 
      and biological molecular mechanism. However, protocols for inducing osteoclast 
      formation in RAW264.7 cells vary considerably between laboratories, hindering the 
      replication of results. Therefore, we tested the influence of culture conditions 
      on osteoclast differentiation, including cell density and receptor activator of 
      nuclear factor kappa-B ligand (RANKL) concentrations with or without macrophage 
      colony-stimulating factors (M-CSF). Tartrate-resistant acid phosphatase (TRAP) 
      staining was used to detect the morphology of osteoclasts. qPCR was used to 
      detect gene expression of osteoclast-specific gene marker cathepsin K (CTSK), 
      osteoclast transcription factors c-Fos and nuclear factor of activated T cells, 
      cytoplasmic 1 (NFATc1). The bone resorption function was evaluated by a scanning 
      electron microscope (SEM). RANKL treatment increased multinucleated osteoclasts 
      formation and increased CTSK, c-Fos and NFATc1 gene expression. Compared with 
      RANKL treatment, M-CSF significantly decreased multinucleated osteoclasts 
      formation, reduced CTSK gene expression and had little effect on c-Fos and NFATc1 
      gene expression. Concerning bone resorption activity, RANKL treatment increased 
      bone resorption pits on bovine bone slices. Significantly higher levels of 
      osteoclastogenesis were observed with RAW264.7-cell density of 2x104 cells/well 
      in 24-well plates. Our results suggest that the addition of 50 ng/ml M-CSF has no 
      positive effect on osteoclastogenesis. RANKL treatment and cell density 
      contribute to osteoclast formation, and the optimal conditions are beneficial 
      when exploring osteoclast function and mechanism.
CI  - Copyright: (c) 2022 Cheng et al. This is an open access article distributed under 
      the terms of the Creative Commons Attribution License, which permits unrestricted 
      use, distribution, and reproduction in any medium, provided the original author 
      and source are credited.
FAU - Cheng, Yin
AU  - Cheng Y
AD  - Institute of Basic Theory, China Academy of Chinese Medical Sciences, Beijing, 
      China.
FAU - Liu, Haixia
AU  - Liu H
AUID- ORCID: 0000-0003-2198-1015
AD  - Institute of Basic Theory, China Academy of Chinese Medical Sciences, Beijing, 
      China.
FAU - Li, Jing
AU  - Li J
AD  - Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School 
      of Basic Medicine Peking Union Medical College, Center of Excellence in Tissue 
      Engineering, Chinese Academy of Medical Sciences, Beijing Key Laboratory of New 
      Drug Development and Clinical Trial of Stem Cell Therapy (BZ0381), Beijing, 
      China.
FAU - Ma, Yujie
AU  - Ma Y
AD  - Institute of Basic Theory, China Academy of Chinese Medical Sciences, Beijing, 
      China.
FAU - Song, Changheng
AU  - Song C
AD  - Institute of Basic Theory, China Academy of Chinese Medical Sciences, Beijing, 
      China.
FAU - Wang, Yuhan
AU  - Wang Y
AD  - Institute of Basic Theory, China Academy of Chinese Medical Sciences, Beijing, 
      China.
FAU - Li, Pei
AU  - Li P
AD  - Institute of Basic Theory, China Academy of Chinese Medical Sciences, Beijing, 
      China.
FAU - Chen, Yanjing
AU  - Chen Y
AD  - Institute of Basic Theory, China Academy of Chinese Medical Sciences, Beijing, 
      China.
FAU - Zhang, Zhiguo
AU  - Zhang Z
AD  - Institute of Basic Theory, China Academy of Chinese Medical Sciences, Beijing, 
      China.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20221117
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 81627-83-0 (Macrophage Colony-Stimulating Factor)
RN  - 0 (NFATC Transcription Factors)
RN  - 0 (Proto-Oncogene Proteins c-fos)
SB  - IM
MH  - Animals
MH  - Cattle
MH  - *Osteogenesis
MH  - Macrophage Colony-Stimulating Factor/pharmacology
MH  - NFATC Transcription Factors/genetics/metabolism
MH  - Cell Differentiation
MH  - *Bone Resorption/genetics/metabolism
MH  - Proto-Oncogene Proteins c-fos/genetics
PMC - PMC9671299
COIS- The authors have declared that no competing interests exist.
EDAT- 2022/11/18 06:00
MHDA- 2022/11/22 06:00
PMCR- 2022/11/17
CRDT- 2022/11/17 14:17
PHST- 2022/02/23 00:00 [received]
PHST- 2022/10/11 00:00 [accepted]
PHST- 2022/11/17 14:17 [entrez]
PHST- 2022/11/18 06:00 [pubmed]
PHST- 2022/11/22 06:00 [medline]
PHST- 2022/11/17 00:00 [pmc-release]
AID - PONE-D-22-05482 [pii]
AID - 10.1371/journal.pone.0277871 [doi]
PST - epublish
SO  - PLoS One. 2022 Nov 17;17(11):e0277871. doi: 10.1371/journal.pone.0277871. 
      eCollection 2022.