Eosinophils were purified from the blood of healthy donors as previously described [11 (link)]. The viability and purity of freshly isolated eosinophils were >99% as analyzed by trypan blue exclusion and Panoptic kit staining, respectively. (250,000/250 μL) were resuspended in RPMI 1640 plus 0.1% ovalbumin (without phenol red) (Sigma, St. Louis, MO, USA) and stimulated with ADP (0.1–100 nM) (Sigma, St. Louis, MO, USA). Treatments with MRS2395 (10 μM) (Sigma, St. Louis, MO, USA), a selective P2Y12R antagonist, or with MRS2179 (10 μM) (Sigma, St. Louis, MO, USA), a selective P2Y1R antagonist, were performed for 15 min prior to stimulation with ADP (100 nM) for 1 h at 37°C. Thereafter, supernatants were collected and stored at −20°C for further EPO quantification. Drugs were diluted in DMSO at a final concentration of <0.01%, which had no effect on eosinophil secretion.
Mrs2179
Manufactured by Merck Group
Sourced in United States
MRS2179 is a laboratory reagent manufactured by Merck Group. It is a small molecule that can be used as a pharmacological tool in research applications. The core function of MRS2179 is to act as an antagonist for the P2Y1 receptor, which is involved in various physiological processes. Detailed information about its intended use or applications is not provided in this factual and unbiased description.
Automatically generated - may contain errors
Lab products found in correlation
Apyrase, by Merck Group (4 mentions)
Adenosine triphosphate (atp), by Merck Group (3 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (3 mentions)
Pd98059, by Merck Group (3 mentions)
Mrs2365, by Merck Group (3 mentions)
2 chloro n6 cyclopentyladenosine ccpa 8 cyclopentyl 1 3 dipropylxanthine dpcpx 2 3 benzoyl 4 benzoyl atp bzatp, by Merck Group (3 mentions)
Periodate oxidized atp oxatp, by Merck Group (3 mentions)
Nf340, by Bio-Techne (3 mentions)
Myr pki, by Enzo Life Sciences (3 mentions)
14 22 amide myristoylated, by Enzo Life Sciences (3 mentions)
N6 ph camp, by Biolog (3 mentions)
Recombinant human transforming growth factor β1 tgf β1, by Merck Group (3 mentions)
Cgs21680, by Bio-Techne (3 mentions)
Zm241385, by Bio-Techne (3 mentions)
Nf546, by Bio-Techne (3 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Adenosine diphosphate (adp), by Merck Group (1 mentions)
Mrs2395, by Merck Group (1 mentions)
Adenosine, by Merck Group (1 mentions)
13 protocols using mrs2179
1
Eosinophil Isolation and ADP Stimulation
2
Pharmacological Compound Preparation
3
Platelet Activation Signaling Pathway
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Thrombin, 2-methylthio-ADP (2-MeSADP), apyrase (type V), MRS2179 (a P2Y1 receptor antagonist [2 (link)]), prostaglandin E1 (PGE1), sodium citrate, and acetylsalicylic acid were purchased from Sigma (USA). Hexapeptides SFLLRN and AYPGKF were custom synthesized by Invitrogen (USA). Anti-phospho-Akt (Ser473) and anti-β-actin antibodies were purchased from Cell Signaling Technology (USA). Horseradish peroxidase-labeled secondary antibody was obtained from Santa Cruz Biotechnology (USA). Bisindolylmaleimide I (GF 109203X) was purchased from Calbiochem (USA). The AR-C69931MX was a gift from AstraZeneca (UK). All other reagents were reagent grade, and deionized water was used throughout.
4
Purinergic Signaling Modulation in Cells
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From Sigma-Aldrich (St. Louis, MO, USA): Adenosine 5′-triphosphate disodium salt hydrate (ATP, A1852), DMEM (D6429), Hanks′ Balanced Salt Solution (HBSS, H4641), HEPES sodium salt (H7006), Monensin (M5273), Nystatin (N6261), Amiloride (PHR1839), Xestospongin C (X2628), MRS-2179 (M3808), U73122 (U6756), Thapsigargin (T9033), Ryanodine (559276), Carbenoxolone (C4790), 10Panx1 (SML2152), Bafilomycin A1 (19-148), Apyrase (A6237), Gap26 (SML3074). From Molecular probes (Eugene, OR, USA): Hoechst 33,342 (H1399), Propidium iodide (P1304MP). From Evrogene (Moscow, Russia): MMLV reverse transcriptase (SK022S), SYBR Green I PCR Master Mix (PK147L); Triethylammonium salt (TNP-ATP, Ann Arbor, MI, USA, 20902). From Thermo Fisher Scientific (Waltham, MA, USA): Fura-2AM (Cat. #F1221), Fetal Bovine Serum (10099141). Selenium nanoparticles (kindly provided by Dr. S.V. Gudkov, Prokhorov General Physics Institute of the Russian Academy of Sciences, Moscow, Russia).
5
Transforming Growth Factor-β1 Signaling Assay
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Recombinant human transforming growth factor-β1 (TGF-β1) was purchased from Millipore (Milan, Italy). PD98059, 3-isobutyl-1-methylxanthine (IBMX), MRS2365, MRS2179, 2-chloro-N6-cyclopentyladenosine (CCPA), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 2′,3′-(benzoyl-4-benzoyl)-ATP (BzATP), and periodate-oxidized ATP (OxATP) were purchased from Sigma-Aldrich. NF546 (4,4′-(carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)carbonylimino))-bis(1,3-xylene-α,α′-diphosphonic acid tetrasodium salt), NF340 [4,4′-(carbonylbis(imino-3,1-(4-methyl-phenylene)carbonylimino))bis(naphthalene-2,6-disulfonic acid) tetrasodium salt], CGS21680, ZM241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol), and 8-CPT-2Me-cAMP (8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate sodium salt) were purchased from Tocris Bioscience (Ellisville, MO, USA). Myr-PKI (protein kinase inhibitor (14-22)-amide myristoylated) was purchased from Enzo Life Sciences (Roma, Italy). 8-CPT-cAMP (8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate) and N6-Ph-cAMP (N6-phenyladenosine-3′,5′-cyclic monophosphate) were purchased from Biolog Life Science Institute (Bremen, Germany). Alexa Fluor 594 phalloidin, 4′,6-diamino-2-phenyl-indole (DAPI), and Slowfade Antifade Reagent were purchased from Life Technologies.
6
Tetramethylpyrazine Inhibits Platelet Activation
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Tetramethylpyrazine (TMP) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Ticagrelor (Tica) was from Selleck (Shanghai, China). Adenosine 5'-diphosphate (ADP) and MRS2179 (P2Y1 receptor antagonist) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Thrombin was from Beijing Solarbio Science & Technology Co., Ltd. (Bejing, China). Dimethyl sulfoxide (DMSO) was from Amresco (Houston, Texas, USA). Antibodies (anti-CD61-APC, anti-CD62P-phycoerythrin-labeled (PE), and anti-GPIIb/IIIa-FITC PAC-1) were purchased from BD Biosciences (San Diego, CA, USA). SQ22536 (an AC inhibitor) and LY294002 (a PI3k inhibitor) were obtained from APExBIO Technology LLC (Houston, Texas, USA). The cyclic AMP enzyme immunoassay (ELISA) kit was from Cayman Chemical (Ann Arbor, MI, USA). The IL-1β and soluble CD40 ligand (sCD40L) ELISA kits were obtained from Elabscience Biotechnology Co., Ltd. (Wuhan, China). The bicinchoninic acid (BCA) protein assay kit was from Beyotime (Shanghai, China). Antibodies against phospho-VASPser157, Akt, phospho-Aktser473, and phospho-AktThr308 were acquired from Cell Signaling Technology (Beverly, MA, USA). Tetramethylpyrazine, SQ22536, and LY294002 were dissolved in DMSO for further use in experiments. DMSO 0.1% was employed as the vehicle.
7
Antagonists of P2 Purinergic Receptors
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P2 receptor antagonists: Pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) tetrasodium salt hydrate (PPADS), MRS2211, MRS2179, MRS2159, A438079, Brilliant Blue G, ATP-2′, 3′-dialdehyde (OxATP), niflumix acid, carbenoxolone disodium salt, and apyrase were purchased from Sigma-Aldrich (St. Louis, MO, United States). NF449, NF023, and Evans Blue tetrasodium salt were purchased from Tocris Bioscience (Abingdon, United Kingdom). Suramin hexasodium salt was purchased from Aladdin (Shanghai, China). Anti-His monoclonal antibody was purchased from EARTHOX Life Sciences (Millbrae, CA, United States). Anti-ETX monoclonal antibody was developed previously by colleagues in our laboratory. HRP-coupled goat anti-mouse IgG antibody was purchased from TransGen Biotech (Beijing, China). The ATP assay kit was purchased from Calbiochem-Merck (Darmstadt, Germany). MTS (3-(4, 5-dimethylthiazol-2-yl) -5(3-carboxymethoxyphenyl) -2-(4-sulfopheny)-2H-tetrazolium, inner salt) was purchased from Promega Corporation (Madison, WI, United States).
8
FAC-MS for Affinity Screening
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The FAC experiment was performed on a chromatographic system (Thermo Finnigan, San Jose, CA, USA). This system included a linear ion trap MS with an ESI ion source, a quaternary gradient Survey or MS pump, and a thermostated column oven Surveyor autosampler. The mobile phase consisted of 10% methanol and 90% ammonium acetate, and a flow rate of 500 μL/min was applied in this experiment at room temperature. Then, the insertion of a post-column mobile phase resulted in only 200 μL/min divert to MS. As a result, mass spectra were generated under constant instrumental conditions in the negative ion mode. During FAC-MS screening runs, the ligand mixture with an equimolar solution was continuously infused. The mixture contained affinity reference compounds in the mobile phase, including cangrelor (Medicines Company, Parsippany, NJ, USA), MRS2179, and uridine diphosphate (UDP) (1 μM, Sigma).
9
Peptide-mediated Cx43 Hemichannel Regulation
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Gap26, TAT-L2 and 10panx1 peptides were obtained from Genscript (New Jersey, USA). HEPES, DMEM, DNAse I, poly-L-lysine, CPP, A74003, MRS2179, brilliant blue G (BBG), oATP, ethidium (Etd) bromide, and probenecid (Prob) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal calf serum (FCS) was obtained from Hyclone (Logan, UT, USA). Penicillin and streptomycin were obtained from Invitrogen (Carlsbad, CA, USA). Normal goat serum (NGS) was purchased from Zymed (San Francisco, CA, USA). Cx43E2, a Cx43 hemichannel antibody to the second extracellular loop was kindly provided by Dr. Jean Jiang, Department of Biochemistry, University of San Antonio, USA (Siller-Jackson et al., 2008 (link)).
10
Transforming Growth Factor-β1 Signaling Assay
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Recombinant human transforming growth factor-β1 (TGF-β1) was purchased from Millipore (Milan, Italy). PD98059, 3-isobutyl-1-methylxanthine (IBMX), MRS2365, MRS2179, 2-chloro-N6-cyclopentyladenosine (CCPA), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 2′,3′-(benzoyl-4-benzoyl)-ATP (BzATP), and periodate-oxidized ATP (OxATP) were purchased from Sigma-Aldrich. NF546 (4,4′-(carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)carbonylimino))-bis(1,3-xylene-α,α′-diphosphonic acid tetrasodium salt), NF340 [4,4′-(carbonylbis(imino-3,1-(4-methyl-phenylene)carbonylimino))bis(naphthalene-2,6-disulfonic acid) tetrasodium salt], CGS21680, ZM241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol), and 8-CPT-2Me-cAMP (8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate sodium salt) were purchased from Tocris Bioscience (Ellisville, MO, USA). Myr-PKI (protein kinase inhibitor (14-22)-amide myristoylated) was purchased from Enzo Life Sciences (Roma, Italy). 8-CPT-cAMP (8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate) and N6-Ph-cAMP (N6-phenyladenosine-3′,5′-cyclic monophosphate) were purchased from Biolog Life Science Institute (Bremen, Germany). Alexa Fluor 594 phalloidin, 4′,6-diamino-2-phenyl-indole (DAPI), and Slowfade Antifade Reagent were purchased from Life Technologies.
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