The soluble human interleukin 6 receptor (IL-6r) was obtained from R&D Systems (Minneapolis, MN). Prostaglandin E2 (PGE2) from was from Cayman Chemical Company (Ann Arbor, MI). 2′,3′-O-(2,4,5-Trinitrophenyl) adenosine-5′-triphosphate tetra(triethylammonium) salt (TNP-ATP) and skepinone-L (skepinone) were obtained from Sigma-Aldrich (St. Louis, MO). IL-6r stock solution was made in sterile phosphate buffered saline (PBS). PGE2 stock solution was made in 100% ethanol. TNP-ATP and skepinone stocks were made in dimethyl sulfoxide. All drugs were diluted to final working concentration in sterile 0.9% saline prior to injection.
Prostaglandin e2 pge2
Manufactured by Cayman Chemical
Sourced in United States
Prostaglandin E2 (PGE2) is a laboratory standard product offered by Cayman Chemical. PGE2 is a biochemical compound that plays a role in various physiological processes. The core function of PGE2 is to serve as a reference material for research and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Isoproterenol, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Santa Cruz Biotechnology (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Indomethacin, by Merck Group (2 mentions)
Okadaic acid, by Bio-Techne (2 mentions)
Penicillin streptomycin, by Welgene (2 mentions)
Fetal bovine serum (fbs), by Welgene (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
λ carrageenan, by Merck Group (2 mentions)
Neutrophil elastase ne, by Worthington (2 mentions)
Compound 48 80, by Merck Group (2 mentions)
Trypsin, by Worthington (2 mentions)
Tram 34, by Bio-Techne (2 mentions)
9 11 dideoxy 9α 11α methanoepoxy prostaglandin f2α u46619, by Cayman Chemical (2 mentions)
Dimethyl sulfoxide (dmso), by Bio-Techne (2 mentions)
1 aminocyclopentane trans 1 3 dicarboxylic acid trans acpd paxilline, by Bio-Techne (2 mentions)
Complete freund adjuvant (cfa), by Merck Group (2 mentions)
Complete freund s adjuvant, by Merck Group (2 mentions)
Ku55933, by Selleck Chemicals (2 mentions)
H1299, by Korean Cell Line Bank (2 mentions)
29 protocols using prostaglandin e2 pge2
1
Preparation of Interleukin-6 Receptor Compounds
2
Cytokine and Inflammatory Mediator Profiling
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Cells were seeded at a density of 105 cells/well in 12-well plates and incubated in 1 ml of growth medium for three days. The culture media were collected, clarified by centrifugation at 1,200 g for ten minutes, supplemented with 2 μg/ml of aprotinin, 17.5 μg/ml of phenylmethylsulphonyl fluoride, 1 μg/ml of pepstatin A, and 50 μg/ml of bacitracin (all from MilliporeSigma, Spain), and stored at -80°C. The levels of interleukin (IL)-6, IL-8, VEGF, and monocyte chemoattractant protein-1 (MCP-1) were determined in cell culture media using BD CBA Flex Sets (BD Biosciences, USA). The data were acquired using a FACSCalibur flow cytometer and analyzed with the FCAP Array Software version 3.0 (BD Biosciences). Human-specific enzyme-linked immunosorbent assay (ELISA) kits were used to measure the levels of OPG (Bender MedSystems GmbH, Austria), RANKL (Biomedica Gruppe, Austria), macrophage colony-stimulating factor (M-CSF) (R&D Systems, Germany), and prostaglandin E2 (PGE2) (Cayman Chemical Company, USA), according to the manufacturer’s instructions.
3
Inflammatory Response Induction in Rats
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LPS, Escherichia coli serotype 0127:B8, (Sigma-Aldrich, catalog #L4516, lot #051M4004) was reconstituted with sterile water. Recombinant rat TNFα (Shenandoah Biolotechnology Inc., catalog #P16599) was reconstituted with sterile water and diluted with a 0.1% BSA solution for prolonged storage. Indomethacin (Sigma-Aldrich, catalog #I7378) was reconstituted with 0.1 mm Na2CO3. Prostaglandin E2 (PGE2; Cayman Chemical, catalog #14 010) was reconstituted with 95% ethanol. Propranolol hydrochloride (Sigma-Aldrich, catalog #P0884) was reconstituted in sterile water. All reagents were further diluted in 0.9% pyrogen-free sterile saline to yield the desired concentration for their administration to the animals.
4
Pharmacological Manipulation of Synaptic Plasticity
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Myristoylated ZIP, pep2m, and scrambled peptide were acquired from Tocris Bioscience; prostaglandin E2 (PGE2) was obtained from Cayman Chemical Company; and nerve growth factor (NGF) was from R&D Systems. ZIP, pep2m and scrambled peptide stock solutions were prepared in sterile 0.9% saline and diluted from this stock solution for injection. PGE2 stock solutions were prepared in 100% ethanol and diluted in sterile saline for injection. Doses used in this study were based on our previous work (Asiedu et al., 2011 ) and other publications (Shi et al., 2001 (link), Pastalkova et al., 2006 (link), Shema et al., 2007 (link)). ZIP is not a selective inhibitor of aPKCs (Lee et al., 2013 (link), Volk et al., 2013 (link)). pep2m interferes with GluA2 binding to NSF and the effect of the peptide is absent in GluA2 KO mice, suggesting it is a specific tool peptide (Shi et al., 2001 (link)).
5
Cultivation of Human Cancer Cell Lines
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Human malignant melanoma cells (SK-MEL-28, male; SK-MEL-2, sex unknown), uterine cervical cancer cells (HeLa, female) and non-small cell lung cancer cells (A549, male; H1299, sex unknown) were obtained from the Korea Cell Line Bank (Seoul, Korea). SiHa human cervical cancer cells (female) were a gift of Dr. HD Youn (Seoul National University, Seoul, Korea). HeLa, SiHa and H1299 cells were grown in Dulbecco's modified Eagle's medium (DMEM), SK-MEL-2 and A549 cells were cultured in RPMI 1640, and SK-MEL-28 cells were cultured in Minimum Essential Medium (MEM). All the media contained 10% fetal bovine serum (Welgene, Gyeongsan, Korea) and 100 units/mL of penicillin/streptomycin (Welgene), and the cells were incubated at 37°C in a 5% CO2 incubator.
Prostaglandin E2 (PGE2) was purchased from Cayman Chemical (Ann Arbor, MI, USA), and okadaic acid was obtained from Tocris Bioscience (Bristol, UK). Isoproterenol, H-89, dimethyl sulfoxide and 4′, 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). N6-phenyladenosine-3′, 5′-cyclic monophosphate (6-Phe-cAMP) was purchased from the Biological Life Science Institute (Bremen, Germany), and bovine serum albumin was obtained from Santa Cruz Biotechnology (Dallas, TX, USA).
Prostaglandin E2 (PGE2) was purchased from Cayman Chemical (Ann Arbor, MI, USA), and okadaic acid was obtained from Tocris Bioscience (Bristol, UK). Isoproterenol, H-89, dimethyl sulfoxide and 4′, 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). N6-phenyladenosine-3′, 5′-cyclic monophosphate (6-Phe-cAMP) was purchased from the Biological Life Science Institute (Bremen, Germany), and bovine serum albumin was obtained from Santa Cruz Biotechnology (Dallas, TX, USA).
6
BDNF Signaling Pathway Analysis
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The BDNF antibodies were purchased from Developmental Studies Hybridoma Bank at the University of Iowa (mouse #9; Iowa City, IA, United States) and Sigma–Aldrich (rabbit; St. Louis, MO, United States). Phospho-eIF4E, GAPDH, and trkB antibodies were obtained from Cell Signaling Technology (Danvers, MA, United States). PAR2 agonist, 2-aminothiazol-4-yl-LIGRL-NH2 (2at-LIGRL), was synthesized as described previously (Boitano et al., 2011 (link)). Human recombinant BDNF was purchased from R&D Systems (Minneapolis, MN, United States). Prostaglandin E2 (PGE2) was purchased from Cayman chemicals (Ann Arbor, MI, United States). All other chemicals were attained from ThermoFisher Scientific (Waltham, MA, United States).
7
Synthesis and In Vitro/Vivo Evaluation of Compounds
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Compound 781 (C781) and 2-aminothiazo-4-yl-LIGRL-NH2 (2AT) were made using solid-phase synthesis, as previously described for 2AT and C781.6 (link),7 (link),20 (link),21 (link),46 ,54 For in vitro studies, neutrophil elastase (NE; cat# LS002274) and trypsin (cat# LS003703) were purchased from Worthington Biochemical Corporation, Lakewood New Jersey. NE was first diluted to 100 mM in Hanks Balanced Saline Solution (HBSS), pH 8.1. All subsequent dilutions of NE, trypsin, C781 or 2AT were in Minimal Essential Medium (MEM), pH 7.4. All experiments were conducted at 37°C and 5% CO2. For in vivo studies, NE was purchased from Elastin Products Company, Inc. (cat# SE563), compound 48/80 from Millipore Sigma (cat# C2313), λ-carrageenan from Sigma Aldrich (cat# 22049), and prostaglandin E2 (PGE2) from Cayman Chemical (cat# 14010). All compounds were dissolved in 0.9% saline. C781 was injected at doses indicated in the text into the intraperitoneal (i.p.) space using a 27G ½” needle. All other compounds were injected into the left hind paw using a glass Hamilton syringe (cat# 80901) and 30G ½” needle.
8
Organoid Culture and Classification
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Isolated crypts were resuspended in Matrigel (Corning) and plated in 48-well culture plates (Corning). After incubation at 37 °C for 15 min, 250 μL of maintenance medium (50% Wnt3a-conditioned medium (ATCC#CRL-2647, Manassas, VA, USA) and 50% of 2× basal medium, supplemented with recombinant human EGF (Sigma-Aldrich), recombinant human noggin (R&D Systems, Minneapolis, MN, USA), recombinant human R-spondin1 (PeproTech, Cranbury, NJ, USA), nicotinamide (Sigma-Aldrich), p160ROCK inhibitor (Selleck Chemicals, Houston, TX, USA), p38 MAP kinase inhibitor (SB202190, Sigma-Aldrich), and Prostaglandin E2 (PGE2, Cayman Chemical, Ann Arbor, MI, USA), were added to the wells. A GSK3 inhibitor (Stemgent, Cambridge, MA, USA) was added to the medium during the first 2 days.
Depending on their shape, organoids can be classified into spheroids and enteroids. Spheroids are defined as round- or oval-shaped organoids with a thin wall composed of a single layer of undifferentiated cells. Enteroids are defined by the presence of visually sharp borders (buddings) along their basolateral (anti-luminal) side or irregularly thickened walls, which consist of all components of epithelial cells [28 (link)] (Figure 10 ).
Depending on their shape, organoids can be classified into spheroids and enteroids. Spheroids are defined as round- or oval-shaped organoids with a thin wall composed of a single layer of undifferentiated cells. Enteroids are defined by the presence of visually sharp borders (buddings) along their basolateral (anti-luminal) side or irregularly thickened walls, which consist of all components of epithelial cells [28 (link)] (
9
Monocyte-Derived Dendritic Cell Generation
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Mo-DCs were prepared from healthy donor-derived PBMCs, as already described [23 (link)]. PBMCs were cultured with CellGro DC Medium (CellGenix GmbH, Freiburg, Germany) at 10 × 106 cells/mL for 2 h after which all cells that had not adhered to the plastic were removed from the culture. Adherent cells were cultured in CellGro DC Medium added with 1000 IU/mL of recombinant human (rh) interleukin (IL)-4 and rh-granulocyte-macrophage colony-stimulating factor (GM-CSF; CellGenix GmbH). On day 7 the culture medium was discarded and the cells were incubated in CellGro DC Medium added with IL-6 (2000 UI/mL), tumor necrosis factor-α (TNFα; 20 ng/mL), IL-1β (20 ng/mL) (all from CellGenix GmbH), and prostaglandin E2 (PGE2; 1 μg/mL) (Cayman Chemical, Ann Arbor, MI, USA). On day 9 mDCs were collected, washed, resuspended in sterile saline solution, and counted under a light microscope to assess their vitality and purity. DCs were cryopreserved in 90% autologous plasma and 10% DMSO solution until use.
10
Compound Sourcing and Preparation for Pharmacological Studies
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