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. 2017 Jul 18;7:5747. doi: 10.1038/s41598-017-06126-x

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Osteoclast-precursor (OCP) differentiation assays. (a–p) 11 week-old male SPF & GF mice were euthanized; bone marrow harvested; hematopoietic progenitor cells (HPCs) isolated. Magnetic cell sorting was applied to separate CD11b^neg HPCs, which were then stimulated in culture (primed with CSF1) to enrich for CD11b^neg osteoclast-precursor (OCP) cells having high osteoclastic potential. CD11b^neg OCP cultures were then stimulated with control (CSF1 alone) or treatment (CSF1 & RANKL) media for 3, 5 and 7 days. Cytomorphometric cellular differentiation endpoints were analyzed in TRAP stained CD11b^neg OCP cultures at day-3, day-5, and day-7 to evaluate cell level alterations in RANKL-induced osteoclast differentiation; TRAP + cell with > 3 nuclei considered an osteoclast. Gene expression studies were carried out in CD11b^neg OCP cultures at day-3 to detect early transcription level alterations in RANKL-stimulated osteoclast differentiation. (a–d) Day-3 TRAP stain assay (n = 4/gp). (a) Representative images (200X) of CD11b^neg OCP cultures stimulated with treatment (CSF1 & RANKL) media for 3 days. (b) N.Oc/Field = number of osteoclasts per field of view. (c) Oc.Ar/Oc = average osteoclast area. (d) N.Nc/Oc = nuclei number per osteoclast. (e–h) Day-3 qRT-PCR gene expression assay (n = 4/gp). (e) Nfatc1 mRNA. (f) Tnfrsf11a mRNA. (g) Csf1r mRNA. (h) Dcstamp mRNA. Relative quantification of mRNA was performed via the comparative C _T method (ΔΔCT); Gapdh was utilized as an internal control gene; data expressed as treatment (CSF1 and RANKL) fold change relative to control (CSF1). (i–l) Day-5 TRAP stain assay (n = 4/gp). (i) Representative images (100×) of CD11b^neg OCP cultures stimulated with treatment (CSF1 & RANKL) media for 5 days. (j) N.Oc/Field. (k) Oc.Ar/Oc. (l) N.Nc/Oc. (m–p) Day-7 TRAP stain assay (n = 4/gp). (m) Representative images (100×) of CD11b^neg OCP cultures stimulated with treatment (CSF1 & RANKL) media for 7 days. (n) N.Oc/Field. (o) Oc.Ar/Oc. (p) N.Nc/Oc. Gene expression assay performed in duplicate (two technical replicate) cultures. TRAP stain assays performed in triplicate (three technical replicate) cultures; three fields of view analyzed per technical replicate culture. n-values represent biological replicates per group. Data reported as mean ± SEM. *p < 0.05 vs. SPF; **p < 0.01 vs. SPF; ***p < 0.001 vs SPF.