osteoclast differentiation medium  (PeproTech)

Journal: Journal of cellular physiology

Article Title: Tributyltin induces distinct effects on cortical and trabecular bone in female C57Bl/6J mice

doi: 10.1002/jcp.26495

Figure Lengend Snippet: Mice were treated as described in Figure 2. a.) Representative images of 5 μm slices of distal femur stained for TRAP (20x magnification)(red shading; n=4 Vh, 5 TBT). b.) Oc/B.Per.: number of TRAP-positive osteoclasts per nm of trabecular perimeter, excluding cortex. Trab. Perimeter: total perimeter of trabecular bone (um). c.) Quantification of serum TRAP by ELISA (n = 12 Vh, 11 TBT 10 mg/kg). Whole humerus bone mRNA expression (n = 12 Vh, 11 TBT 10 mg/kg) of d.) Osteoclast differentiation markers Nfatc1, Apc5 and Ctsk, and e.) Intercellular communication proteins Rankl/Opg (osteoblast to osteoclast), Ct1 and Sema4d (osteoclast to osteoblast). Data are presented as mean ± SE. *p < 0.05 **p < 0.01, Mann-Whitney.

Article Snippet: For resorption assays, cells were collected from the differentiating osteoclast cultures, counted, plated (40,000 per well in 800 μl osteoclast differentiation medium) in 24-well Corning Osteo-Assay plates (CLS3987, Sigma Aldrich) and re-treated with Vh or TBT.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Expressing, MANN-WHITNEY

Journal: Journal of cellular physiology

Article Title: Tributyltin induces distinct effects on cortical and trabecular bone in female C57Bl/6J mice

doi: 10.1002/jcp.26495

Figure Lengend Snippet: Primary bone marrow macrophages were isolated from female C57BL/6J mice, induced to differentiate to osteoclasts with M-CSF and RANKL (see Methods), and after 24 hrs treated with Vh (DMSO), TBT (20, 50, or 80 nM), rosiglitazone (Rosi, 500 nM, PPARγ agonist), LG100268 (LG268, RXR agonist 1 μM), or T0101317 (T317, LXRα/β agonist, 1 μM) for a total of 6 days of differentiation. a.) mRNA expression of osteoclast differentiation markers. n=10–17 independent cultures. Data are presented as mean ± SE. *p < 0.05, **p < 0.01, ***p < 0.001 compared to Vh, one-way ANOVA (Dunnett’s). TBT treatments were compared to Vh separately from control comparisons. b.) Representative images of TRAP-positive, multinucleated (3 or more nuclei) cells (Scale bar = 400 μm) n=10 independent cultures. c.) Representative images of von Kossa stained mineral surface after 3 days of incubation with differentiated osteoclasts. n=5 independent cultures.

Article Snippet: For resorption assays, cells were collected from the differentiating osteoclast cultures, counted, plated (40,000 per well in 800 μl osteoclast differentiation medium) in 24-well Corning Osteo-Assay plates (CLS3987, Sigma Aldrich) and re-treated with Vh or TBT.

Techniques: Isolation, Expressing, Control, Staining, Incubation

Journal: Journal of cellular physiology

Article Title: Tributyltin induces distinct effects on cortical and trabecular bone in female C57Bl/6J mice

doi: 10.1002/jcp.26495

Figure Lengend Snippet: a.) Mice were treated as described in Figure 2. Whole humerus bone mRNA expression of LXR-dependent genes Abca1 and Srebp1c. Data are presented as mean ± SE. n = 11–12 individual mice. **p<0.01, Mann-Whitney b.) Osteoclast cultures were prepared as described in Figure 5 and treated with Vh (DMSO), TBT (20, 50, or 80 nM), rosiglitazone (Rosi, 500 nM, PPARγ agonist), LG100268 (LG268, RXR agonist 1 μM), or T0101317 (T317, LXRα/β agonist, 1 μM) and analyzed for mRNA expression Abca1 and Srebp1c. n=10–17 independent cultures. Data are presented as mean ± SE. *p<0.05, **p<0.01, ***p<0.001 compared to Vh, one-way ANOVA (Dunnett’s). TBT treatments were compared to Vh separately from control comparisons. c.) Primary bone marrow macrophages were isolated from male and female LXRα +/+, +/− and −/− mice, induced to differentiate into osteoclasts with M-CSF and RANKL, treated after 24 hrs with Vh (DMSO) or TBT (50 nM) for 4 days, and analyzed for mRNA expression of Ctsk. n=4 independent cultures. Data are presented as mean ± SE. Two-way ANOVA. d.) Osteoclast cultures were prepared as described in b and treated with Vh (DMSO) or TBT (50 nM) with or without GSK2033 (GSK, LXR antagonist, 0.8 μM) and analyzed for mRNA expression Abca1, Srebp1c and Ctsk. n=5 independent cultures. Data are presented as mean ± SE. Two-way ANOVA.

Article Snippet: For resorption assays, cells were collected from the differentiating osteoclast cultures, counted, plated (40,000 per well in 800 μl osteoclast differentiation medium) in 24-well Corning Osteo-Assay plates (CLS3987, Sigma Aldrich) and re-treated with Vh or TBT.

Techniques: Expressing, MANN-WHITNEY, Control, Isolation

Journal: Journal of cellular physiology

Article Title: Tributyltin induces distinct effects on cortical and trabecular bone in female C57Bl/6J mice

doi: 10.1002/jcp.26495

Figure Lengend Snippet: a.) Mice were treated as described in Figure 2. Whole humerus bone mRNA expression the RXR-dependent gene Mafb. Data are presented as mean ± SE. n = 11–12 individual mice. **p<0.01, Mann-Whitney b.) Osteoclast cultures were prepared as described in Figure 5 and treated with Vh (DMSO), TBT (20, 50, or 80 nM), rosiglitazone (500 nM), LG100268 (LG268, 1 μM), or T0101317 (T317, LXRα/β agonist, 1 μM) and analyzed for mRNA expression of Mafb. Data are presented as mean ± SE. n=10–17 independent cultures. *p<0.05, **p<0.01, *** p<0.001 compared to Vh, one-way ANOVA (Dunnett’s). TBT treatments were compared to Vh separately from control comparisons. Osteoclast cultures were prepared as described in b and treated with Vh (DMSO) or TBT (50 nM) with or without HX531 (RXR antagonist, 1 μM) and analyzed for mRNA expression Mafb and Ctsk. n=5 independent cultures. Data are presented as mean ± SE. Two-way ANOVA.

Article Snippet: For resorption assays, cells were collected from the differentiating osteoclast cultures, counted, plated (40,000 per well in 800 μl osteoclast differentiation medium) in 24-well Corning Osteo-Assay plates (CLS3987, Sigma Aldrich) and re-treated with Vh or TBT.

Techniques: Expressing, MANN-WHITNEY, Control

Journal: Stem Cells International

Article Title: Mesenchymal Progenitors Derived from Different Locations in Long Bones Display Diverse Characteristics

doi: 10.1155/2019/5037578

Figure Lengend Snippet: Conditioned medium from E-MPs promoted osteoclast formation. (a) TRAP staining was used to detect osteoclast formation from osteoclast precursors, after culturing in osteoclastic differentiation medium with CM from different MPs for 7 days. Scale bar, 200 μ m. OCi: osteoclast induction. (b) TRAP-positive cells (nuclei ≥ 3) were counted. (c) The mRNA levels of osteoclast marker genes were detected by qRT-PCR. (d) The expression of several paracrine genes which were related to osteoclast formation was evaluated in the three MP groups. Data was shown as mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Osteoclast precursors were incubated with osteoclastic differentiation medium ( α -MEM with 10% FBS, 50 ng/ml M-CSF, and 100 ng/ml RANKL (PeproTech)) for 7 days to induce maturation into osteoclasts.

Techniques: Staining, Marker, Quantitative RT-PCR, Expressing