Murine bone marrow macrophages were isolated from bone marrow flushed tibiae and femurs of WT and Rspo3+/- mice at 6–8 wk-old as described previously (Chen et al., 2019 (link)). Briefly, cells were cultured in complete α-MEM with 30 ng/ml macrophage colony-stimulating factor M-CSF (R&D system, Minneapolis, MN, USA) in suspension culture dish to which stromal cells and lymphoid cells cannot adhere, at 37 °C for 2–3 days. For osteoclast generation, cells were cultured in 30 ng/ml M-CSF and 10 ng/ml RANKL (R&D systems, Minneapolis, MN, USA). For co-culture experiments, mouse calvarial osteoblasts were isolated from newborn WT and Rspo3+/- as previously reported (Movérare-Skrtic et al., 2014 (link); Chen et al., 2019 (link)) and seeded in 96-well plates (2.000 cells/well) in complete osteogenic α-MEM containing 100 nM Vitamin D3 and 1 µM prostaglandin E2 (Enzo Life Science, Farmingdale, NY, USA). After 3 days, 10,000 BMM from WT and Rspo3+/- mice at 6–8 week-old mice were added per well and cocultured for 9 days in complete osteogenic α-MEM. Tartrate-resistant acid phosphatase (TRAP) staining was performed to evaluate the number of osteoclasts according to the manufacture’s protocol (Sigma-Aldrich, St. Louis, MO, USA).
Prostaglandin e2
Manufactured by Enzo Life Sciences
Sourced in United States
Prostaglandin E2 is a key prostanoid that regulates various physiological and pathological processes. It is a primary product of the cyclooxygenase pathway and plays a critical role in inflammation, immune regulation, and other biological functions.
Automatically generated - may contain errors
Lab products found in correlation
Macrophage colony stimulating factor m csf, by R&D Systems (2 mentions)
Complete osteogenic α mem, by Enzo Life Sciences (2 mentions)
Vitamin d3, by Enzo Life Sciences (2 mentions)
M csf, by R&D Systems (2 mentions)
Rankl, by R&D Systems (2 mentions)
Duoset elisa, by R&D Systems (1 mentions)
Naltrindole, by Merck Group (1 mentions)
Clocinnamox, by Merck Group (1 mentions)
Naltrindole, by Bio-Techne (1 mentions)
Carrageenan, by Merck Group (1 mentions)
Nor binaltorphimine dihydrochloride, by Merck Group (1 mentions)
Am251, by Bio-Techne (1 mentions)
Am630, by Bio-Techne (1 mentions)
Am630, by Merck Group (1 mentions)
Nor bni, by Merck Group (1 mentions)
Clocinnamox, by Bio-Techne (1 mentions)
Am251, by Merck Group (1 mentions)
Naloxone, by Merck Group (1 mentions)
Platelet derived growth factor, by R&D Systems (1 mentions)
Vetscan hm2 hematology analyzer, by Abaxis (1 mentions)
5 protocols using prostaglandin e2
1
Murine Bone Marrow Macrophage Isolation and Osteoclast Generation
2
Cytokine and Mediator ELISA Profiling
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Cytokines and mediators in cell supernatants were detected using ELISA according to the manufactor’s instructions. Kits used for various ELISA are as follows: Human IL-1β, IL-6, IL-10, IL-12p70, IL-18, TNF (all BD OptEIA); human IL-1a/IL-1F1, IL-23, IL-18bp, IL-1RA, osteopontin (all DuoSet ELISA, R&D); Prostaglandin E2 (Enzo Life Sciences), and thrombin-cleaved osteopontin (Immuno-Biological Laboratories).
3
Analgesic Compounds: Purification and Preparation
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The following drugs and chemicals were used: δ-CNTX-Pn1a was purified by a combination of preparative reverse phase HPLC (RP-HPLC), ion exchange HPLC and analytical reverse phase HPLC as previously described [52 (link)]. µ-Conotoxin MVIIA was purchased from Latoxan (Valence, France). Carrageenan (Sigma, St Louis, MO, USA), Prostaglandin E2 (Enzo Life Sciences, Farmingdale, NY, USA), AM251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; Tocris, Pittsburg, PA, USA), AM630 (6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl(4-ethoxyphenyl) methanone Tocris, Pittsburgh, PA, USA), Naloxone (Sigma, St. Louis, MO, USA), Clocinnamox (Tocris, Pittsburgh, PA, USA), Naltrindole (Tocris, Pittsburgh, PA, USA), Nor-BNI (Nor-Binaltorphimine dihydrochloride; Sigma, St. Louis, MO, USA) were dissolved as follows: PGE2 (2% ethanol in saline); AM251 and AM630 (12% DMSO in saline); Carrageenan, δ-CNTX-Pn1a, µ-Conotoxin MVIIA (MVIIA), Naloxone, Clocinnamox, Naltrindole and Nor-BNI (saline).
4
Murine Bone Marrow Macrophage Osteoclastogenesis
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Murine bone marrow macrophages were isolated from bone marrow flushed tibiae and femurs of wt and Rspo3 +/-mice at 6 to 8-wk-old as described previously (46) (link). Briefly, cells were cultured in complete α-MEM with 30 ng/ml macrophage colony-stimulating factor M-CSF (R&D system, Minneapolis, MN, USA) in suspension culture dish to which stromal cells and lymphoid cells cannot adhere, at 37 °C for 2-3 days. For osteoclast generation, cells were cultured in 30 ng/ml M-CSF and 10 ng/ml RANKL (R&D systems, Minneapolis, MN, USA). For co-culture experiments, mouse calvarial osteoblasts were isolated from newborn wt and Rspo3 +/-as previously reported (46, (link)47) (link) and seeded in 96-well plates
(2.000 cells/well) in complete osteogenic α-MEM containing 100 nM Vitamin D3 and 1 µM prostaglandin E2 (Enzo Life Science, Farmingdale, NY, USA). After 3 days, 10,000 BMM from wt and Rspo3 +/-mice at 6 to 8-week-old mice were added per well and cocultured for 9 days in complete osteogenic α-MEM. Tartrate-resistant acid phosphatase (TRAP) staining was performed to evaluate the number of osteoclasts according to the manufacture's protocol (Sigma-Aldrich, St. Louis, MO, USA).
(2.000 cells/well) in complete osteogenic α-MEM containing 100 nM Vitamin D3 and 1 µM prostaglandin E2 (Enzo Life Science, Farmingdale, NY, USA). After 3 days, 10,000 BMM from wt and Rspo3 +/-mice at 6 to 8-week-old mice were added per well and cocultured for 9 days in complete osteogenic α-MEM. Tartrate-resistant acid phosphatase (TRAP) staining was performed to evaluate the number of osteoclasts according to the manufacture's protocol (Sigma-Aldrich, St. Louis, MO, USA).
5
Platelet Isolation and Cytokine Quantification
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This study was approved by the institutional review board at Osaka University Hospital. For in vitro experiments, platelets were isolated from the venous blood of one healthy volunteer. The platelet preparation method was adapted from Ford et al. [14] . Five vol of OptiPrep TM was diluted with 22 vol of Solution B [0.85 % (w/v) NaCl, 20 mM Hepes-NaOH, pH 7.4, 1 mM EDTA] to produce a 1.063 g/ml density barrier. In a 15-ml centrifuge tube, 5 ml of blood was layered over 5 ml of the 1.063 g/ml solution and centrifuged at 350 g for 15 min at 20 °C in a swingingbucket rotor, and the rotor was allowed to decelerate without braking. The platelet-containing band was harvested from the broad turbid band below the interface. Isolated platelets were counted in a Vetscan HM2 Hematology Analyzer (Abaxis) and suspended in RPMI 1640 for concentration adjustment. Cytokines in the culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Platelet-derived growth factor (R&D Systems, Minneapolis, MN, USA), transforming growth factor-b (R&D Systems, Minneapolis, MN, USA), and prostaglandin E2 (ENZO Life Science, Famingdale, NY, USA) were measured using an ELISA kit, and interleukin-6 was measured by LSI Medience Corp. (Tokyo, Japan).
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