Stimulation (18 h) prior to migration assays (see below) was performed at 1×106 cells per mL by adding 1 nM of IL-3 (Peprotech), 1 µM of PGD2, 3-(3-Cyclohexyl-3-hydroxypropyl)-2,5-dioxo-(R*,S*)-(±)-4-imidazolineheptanioc acid (BW245c) or 13,14-dihydro-15-keto Prostaglandin D2 (DK-PGD2) (Cayman Chemicals) or 5 ng/mL of anti-IgE (Thermo Scientific). For CXCR4 expression modulation purified human basophils or mouse splenocytes were resuspended in RPMI containing 0.1% BSA±: vehicle, 50 nM CCL3 or CCL5 or CXCL2 (BioLegend), 1 µM Laropiprant, CAY10471, DK-PGD2, BW245c, Prostaglandin D Synthase (hematopoietic-type) Inhibitor I (H-PGDS inhibitor), 1 or 10 µMPGD2 (Cayman Chemicals), 50 µM 2′,5′-dideoxyadenosine (ddAde), 10 µM Forskolin and N6,2′-O-dibutyryl-adenosine 3′:5′-cyclic monophosphate (db-cAMP) (Sigma-Aldrich) or at concentrations specified in figure legends. Cells were incubated for 4 h (unless otherwise specified in figure legends) at 37 °C and 5% CO2 and surface expression of the indicated markers were assessed by flow cytometry.
Bw245c
Manufactured by Cayman Chemical
Sourced in United States
BW245C is a laboratory instrument designed for centrifugal separation. It features a temperature-controlled environment and a range of rotor options to accommodate various sample types and volumes.
Automatically generated - may contain errors
Lab products found in correlation
Dk pgd2, by Cayman Chemical (5 mentions)
Cay10471, by Cayman Chemical (5 mentions)
Mk0524, by Cayman Chemical (4 mentions)
Butaprost, by Cayman Chemical (3 mentions)
Bwa868c, by Cayman Chemical (3 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
15 r 15 methyl pgd2, by Cayman Chemical (2 mentions)
Iloprost, by Cayman Chemical (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
U46619, by Cayman Chemical (2 mentions)
Recombinant human insulin, by Merck Group (1 mentions)
Fatty acid free bovine serum albumin, by Merck Group (1 mentions)
3 isobutyl 1 methylxanthine, by Fujifilm (1 mentions)
L ascorbic acid phosphate magnesium salt n hydrate, by Fujifilm (1 mentions)
Triglyceride e test kit, by Fujifilm (1 mentions)
Mre 269, by Cayman Chemical (1 mentions)
Pcr master mix, by Promega (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Niacin, by Merck Group (1 mentions)
Il 13, by Thermo Fisher Scientific (1 mentions)
14 protocols using bw245c
1
Basophil Migration Assay Optimization
2
Adipocyte Differentiation Culture Protocol
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The following were obtained from the respective suppliers: Dulbecco’s modified Eagle medium containing 25 mM HEPES, penicillin G potassium salt, streptomycin sulfate, dexamethasone, fatty acid-free bovine serum albumin, and recombinant human insulin (Sigma-Aldrich Corp., St. Louis, MO, USA); L-ascorbic acid phosphate magnesium salt n-hydrate, 3-isobutyl-1-methylxanthine (IBMX), and Triglyceride E-Test Kits (Wako Pure Chemical Industries Ltd., Osaka, Japan); fetal bovine serum (FBS) (MP Biomedicals, Solon, OH, USA); PGD2, 11d-11m-PGD2, MRE-269, BW245C, and 15R-15-methyl-PGD2 (15R-15m-PGD2) (Cayman Chemical (Ann Arbor, MI, USA); M-MLV reverse transcriptase (Ribonuclease H minus, point mutant) and polymerase chain reaction (PCR) Master Mix (Promega Corp., Madison, WI, USA).
3
Dextran Sulfate Sodium Colitis Model
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Dextran sulfate sodium (DSS) was purchased from MP Biomedicals (LLC, Santa Ana, California, USA). TNBS, LPS, Evan's blue, and niacin were purchased from Sigma Chemical Company (Sigma‐Aldrich, St. Louis, MO, USA). PGD2 and BW245C were obtained from Cayman Chemical Company (Cayman Chemical, Ann Arbor, MI, USA). Percoll solution was from Biosharp (Biosharp, Hefei, China). IL‐13 was purchased from Peprotech (Peprotech, Rocky Hill, USA). TdT fluorescence in situ Apoptosis Detection kit was from Yeasen Biological Technology (Yeasen, Shanghai, China). Annexin V‐FITC Apoptosis Detection Assay kit was obtained from Dojindo Laboratories (Dojindo, Shanghai, China).
4
Immune Cell Activation and Quantification
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PGD2, PGE2, BW245C (DP1-specific agonist) and DKPGD2 (DP2-specific agonist) were purchased from Cayman Chemical (Ann Arbor, MI) and Rp-8Br-cAMP was purchased from Calbiochem (Darmstadt, Germany). The following monoclonal antibodies were used: anti-human CD3-PerCP (SK7), anti-human CD3-APC (UCHT1), anti-human CD19-APC (HIB19), anti-human CD16-PE (3G8), anti-human CD56-PE (NCAM16.2), anti-human CD107a-FITC (H4A3), anti-human IFN-γ-FITC (25723.11), anti-human DNAM-1-PE (DX11), anti-human NKp46-PE (9E2), anti-human NKp44-PE (p44-8), anti-mouse CD3ε-PerCP (145-2C11), anti-mouse NKp46-PE (29A1.4), anti-mouse CD107a-FITC (1D4B), anti-mouse IFN-γ-FITC (XMG1.3), and anti-mouse CD16/CD32 (mouse Fc Block; 2.4G2) from BD Biosciences (San Jose, CA); anti-human 2B4-PE (C1.7) and anti-human NKp30-PE (Z25) from Beckman Coulter; anti-human NKG2C-PE (134591) and anti-human NKG2D-PE (149810) from R&D Systems; anti-human perforin-Alexa 647 (dG9) and anti-human granzyme B-Alexa 647 (GB11) from BioLegend. Annexin V-FITC and 7-AAD were from BD Biosciences.
5
Eosinophil Migration Assays with EP and DP Agonists
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Migration assays were performed with peripheral blood eosinophils in 96-well Transwell plates with 5-µm membrane inserts (Corning Inc., Lowell, MA, USA). Eosinophils from healthy donors were incubated with the following compounds: EP4 agonist ONO-AE1-329 (100 nM) or EP4 antagonist ONO-AE3-208 (100 nM; ONO compounds were a generous gift from ONO Pharmaceutical, Osaka, Japan), EP2 agonist butaprost (100 and 300 nM; Cayman Chemicals, Ann Arbor, MI, USA), DP1 agonist BW245C (100 nM), and DP1 antagonist MK0524 (1 µM; Cayman Chemicals). Thereafter, a suspension of 5 × 104 eosinophils was placed in the upper well. Supernatant from the esophageal epithelial cell culture was added into the bottom well as a chemoattractant. Eosinophils were allowed to migrate for 1 h at 37 °C and 5% CO2 in a humidified incubator and evaluated by flow cytometry. Each migration experiment was performed in triplicates. The average number of migrated cells was determined from at least five independent experiments.
6
Pharmacological Modulation of Lipid Mediators
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Protectin DX, NS-398(selective COX-2 inhibitor), AT-56 (L-PGDS inhibitor), BW245C (DP1 receptor agonist), 15(R)-15-methyl-PGD2(CRTH2/DP2 receptor agonist), MK-0524(DP1 receptor antagonist) and CAY-10471(CRTH2/DP2 receptor antagonist) were obtained from Cayman Chemical (Ann Arbor, MI, USA), Lipopolysaccharide (Escherichia coli O55: B5), BOC-2 (ALX/FPR2 receptor inhibitor) were purchased from Biomol/Enzo Life Sciences (Farmingdale, NY, USA).
7
Purification and Analysis of Prostaglandin Analogs
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Proton NMR spectra were recorded
in solvent in CDCl3 on a Varian Inova 400 (400 MHz) instrument.
Thin layer chromatography was performed on precoated, aluminum-backed
plates (silica gel 60 F254, 0.25 mm thickness) from EM
Science and was visualized by UV lamp. Column chromatography was performed
with silica gel cartridges on Teledyne-ISCO machine. An Agilent LCMS
instrument was used to measure purity of the products. Elemental analyses
were performed by Atlantic Microlab Inc. (Norcross, GA). Chemicals
and drugs PGE2, BW245C, iloprost, and rolipram were purchased
from Cayman Chemical.
in solvent in CDCl3 on a Varian Inova 400 (400 MHz) instrument.
Thin layer chromatography was performed on precoated, aluminum-backed
plates (silica gel 60 F254, 0.25 mm thickness) from EM
Science and was visualized by UV lamp. Column chromatography was performed
with silica gel cartridges on Teledyne-ISCO machine. An Agilent LCMS
instrument was used to measure purity of the products. Elemental analyses
were performed by Atlantic Microlab Inc. (Norcross, GA). Chemicals
and drugs PGE2, BW245C, iloprost, and rolipram were purchased
from Cayman Chemical.
8
Prostaglandin Receptor Signaling Assays
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All chemicals were purchased from Wako (Osaka, Japan) unless stated otherwise. PGD2, 15d-PGJ2, BW 245C, 15(R)-15-methyl PGD2, MK0524, CAY10471, and GW9662 were obtained from Cayman Chemical (Ann Arbor, MI, USA). These compounds were dissolved in dimethyl sulfoxide (DMSO; Sigma–Aldrich, St. Louis, MO, USA). The primary antibodies used were against DP1 (Cayman Chemical, Ann Arbor, MI, USA, diluted 1:1000), DP2 (Novus Biologicals, Centennial CO, USA, diluted 1:1000), Islet-1 (Cell Signaling Technology, Danvers, MA, USA, diluted 1:1000), and β-actin (Sigma-Aldrich, St. Louis, MO, USA, diluted 1:1000).
9
Synthesis and Preparation of Prostanoid Compounds
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The compounds PGN‐9856, PGN‐9856‐isopropyl ester, PGN‐9858, PGN‐9862, PGN‐9863, CP‐533536 and PF‐04217329 were synthesized by Target Molecules (Southampton, England). Their structures are shown in Figure 1 . Butaprost, PGD2, PGE2, iloprost, carbaprostacyclin, 17‐phenyl‐PGF2α, U‐46619 and BW 245C were purchased from Cayman Chemical (Kalamazoo, MI, USA). The anti‐CD3 monoclonal antibody (clone: OKT3) was supplied by Janssen‐Cilag Ltd. (High Wycombe, UK); LPS from Escherichia coli by Sigma Aldrich Ltd. (Poole, UK), and the proparacaine was supplied by Allergan Inc. (Irvine, CA). For ocular studies, PGN‐9856i and PF‐04217329 were prepared as 0.1% (1 mg·mL−1) suspensions in 10 mM Tris/1% polysorbate 80.
10
Hippocampal Neuron Protection Study
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The hippocampal neurons were divided into groups: the control group, the normal solvent group (300 μM of maltol), the normal intervention group (10−5 M BW245C, BWA868C, DK-PGD2, CAY10471) (Cayman, USA), the model group (100 μM Al(malt)3), the model solvent group (Al(malt)3 +10−3 M DMSO), and the model intervention group (Al(malt)3 + 10−5 M BW245C, BWA868C, DK-PGD2, CAY10471).
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