Bay 60 6583, by Bio-Techne - Product details

1

Isolation and Purification of Kupffer Cells

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Kupffer cells were isolated from mice liver. In brief, under deep anesthesia using pentobarbital (50 mg/kg, i.p.), the liver was perfused in situ with 10 ml of PBS (37°C) in the nonrecirculating fashion to drive out RBCs and subsequently perfused with 10 ml of RPMI 1640 (Hyclone) containing 0.1% collagenase (type IV; Sigma-Aldrich). The liver was removed, minced, and filtered through the mesh. The samples were suspended in RPMI 1640 and centrifuged at 300 × g for 5 min at 4°C; the cell sediments were resuspended with RPMI 1640 and centrifuged at 50 × g for 1 min. The top aqueous phase containing mainly nonparenchymal cells was reserved. To further purify the cells, cells were seeded on the culture plate and incubated for 2–3 h in a humidified incubator (37°C, 5% CO₂). Nonadherent cells were removed from the dish by washing with PBS, and the adherent cells were Kupffer cells (16 (link)). For the experiment with BAY 60–6583 (Tocris Bioscience), the isolated Kupffer cells from G2A^+/+ and G2A^−/−mice were preincubated with LPS (1 μg/ml) for 24 h, and then LPS was washed out, and the cells were exposed to BAY 60–6583 (1 μM) for 10 min, and the intracellular cAMP levels were measured.

Li H.M., Jang J.H., Jung J.S., Shin J., Park C.O., Kim Y.J., Ahn W.G., Nam J.S., Hong C.W., Lee J., Jung Y.J., Chen J.F., Ravid K., Lee H.T., Huh W.K., Kabarowski J.H, & Song D.K. (2018). G2A Protects Mice against Sepsis by Modulating Kupffer Cell Activation: Cooperativity with Adenosine Receptor 2b. Journal of immunology (Baltimore, Md. : 1950), 202(2), 527-538.

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2

Binding Assays for Adenosine Receptors

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PSB1115 (4-(2,3,6,7-Tetrahydro-2,6-dioxo-1-propyl-1H-purin-8-yl)-benzenesulfonic acid), ZM241385 (4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol), BAY60–6583 (2-[[6-Amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]-2-pyridinyl]thio]-acetamide), and CGS21680 (4-[2-[[6-Amino-9-(N-ethyl-β-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride) were purchased from Tocris Bioscience (Minneapolis, MN). Nitrobenzylthioinosine (NBTI), dipyridamole, αβ-methylene ADP, and 5’-deoxycoformycin and Ac-YVAD-cmk were purchased from Sigma Aldrich (St. Louis, MO).

Chambers E.D., White A., Vang A., Wang Z., Ayala A., Weng T., Blackburn M., Choudhary G., Rounds S, & Lu Q. (2019). Blockade of equilibrative nucleoside transporter 1/2 protects against pseudomonas aeruginosa-induced acute lung injury and NLRP3 inflammasome activation. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(1), 1516-1531.

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3

Nebulized Adora2b Agonist for ALI

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For the administration of the highly specific agonist for Adora2b, BAY 60-6583 (1 mg/kg body weight; Tocris Bioscience, Bristol, UK) we used an Aeroneb Lab ultrasound nebulizer (Aerogen, Dangan, Galway, Ireland) suitable for alveolar deposition (particle size: Volumetric Mean Diameter (VMD): 2,5–4,0 μm). The nebulizer unit was placed at the inspiratory side of the ventilator and was triggered during inspiration only. The aerosol treatment (BAY 60-6583 or vehicle) was given during the 3 h of pressure-controlled ventilation, otherwise the animals were treated as described under “Two-hit ALI model”.

Hoegl S., Brodsky K.S., Blackburn M.R., Karmouty-Quintana H., Zwissler B, & Eltzschig H.K. (2015). Alveolar-epithelial A2B adenosine receptors in pulmonary protection during acute lung injury. Journal of immunology (Baltimore, Md. : 1950), 195(4), 1815-1824.

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4

In Vivo Administration of BAY 60-6583

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BAY 60-6583 (Tocris Bioscience, Minneapolis, MN) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO). For in vivo administration, fresh solutions of BAY 60-6583 (10%, v/v), 0.9% sodium chloride (NaCl; 50%, v/v; Hospira), and polyethylene glycol 400 (PEG 400; 40%, v/v; Thermo Fisher Scientific, Hampton, NH) were mixed and sterile-filtered through 0.22-μm-pore filter disk, and 100 μl of solution was injected intraperitoneally at mouse weight (1 mg/kg), after 8 weeks of ovariectomy surgery, once every 2 days. A solution comprising DMSO (10%, v/v), 0.9% NaCl (50%, v/v), and PEG 400 (40%, v/v) was injected in animals as vehicle control.

Shih Y.R., Liu M., Kwon S.K., Iida M., Gong Y., Sangaj N, & Varghese S. (2019). Dysregulation of ectonucleotidase-mediated extracellular adenosine during postmenopausal bone loss. Science Advances, 5(8), eaax1387.

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In Vitro Osteoclastogenesis Assay

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Isolation of bone marrow precursors and the in vitro osteoclastogenesis experiment were performed as described previously (Choi et al., 2013 ). In brief, bone marrow cells isolated from femurs of 4–6 week-old C57BL/6 male mice were cultured in the presence of M-CSF (20 ng/ml, R&D Systems) for 3 days. After washing out the non-adherent cells, the adherent cells were used as BMMs. For osteoclast formation, the isolated preosteoclasts were further cultured in the presence of 200 ng/ml of RANKL (Huh et al., 2016 (link)), 30 ng/ml of M-CSF and/or BAY 60-6583 (Tocris). After 3 days, the cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP) using a TRAP staining kit (Sigma). The cells were observed using a Zeiss Axiovert 200 microscope and images were obtained with an AxioCam HR (Carl Zeiss) equipped with Axio Vision 3.1 software (Carl Zeiss). TRAP-positive multinucleated cells (TRAP⁺ MNCs) larger than 100 μm in diameter containing more than 20 nuclei and TRAP⁺ mononuclear cells were counted, and the number was presented as relative percentage (%).

Kim B.H., Oh J.H, & Lee N.K. (2017). The Inactivation of ERK1/2, p38 and NF-kB Is Involved in the Down-Regulation of Osteoclastogenesis and Function by A2B Adenosine Receptor Stimulation. Molecules and Cells, 40(10), 752-760.

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6

Sprague Dawley Rat Biochemical Assay

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Male Sprague Dawley (SD) rats were obtained from Charles River. Culture media and additives were obtained from Invitrogen. All chemicals were obtained from Sigma unless otherwise stated. BAY60-6583 was from Tocris. Antibodies to YAP, phospho-YAPS127, phospho-YAPS397, TAZ, pan-TEAD and phospho-Retinoblastoma protein were from Cell Signalling Technologies. Anti-BrDU antibody was from Sigma.

Kimura T.E., Duggirala A., Smith M.C., White S., Sala-Newby G.B., Newby A.C, & Bond M. (2016). The Hippo pathway mediates inhibition of vascular smooth muscle cell proliferation by cAMP. Journal of Molecular and Cellular Cardiology, 90, 1-10.

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7

Adora2b Deficiency and DSS-Induced Colitis

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Adora2b-deficient mice,^{8 (link)} mice with tissue-specific Adora2b deletion (for more details see Supplementary Materials), matched genetic controls, or C57BL/6 mice were used in DSS studies (3–4.5%), as described previously.^{13 (link),44 (link),45 (link)} Adora2b agonist (BAY 60-6583; Tocris Bioscience, Bristol, UK) or vehicle (30% SolutolHS15 in 0.9% saline; BASF, Florham Park, NJ) was administered by subcutaneous osmotic pump (Alzet; DURECT Corporation, Cupertino, CA) at 1.2–1.25 mg kg⁻¹ mouse per day.

Aherne C., Saeedi B., Collins C., Masterson J., McNamee E., Perrenoud L., Rapp C., Curtis V., Bayless A., Fletcher A., Glover L., Evans C., Jedlicka P., Furuta G., de Zoeten E., Colgan S, & Eltzschig H. (2015). Epithelial-specific A2B adenosine receptor signaling protects the colonic epithelial barrier during acute colitis. Mucosal immunology, 8(6), 1324-1338.

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8

Adenosine Receptor Ligands and Analogs

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The adenosine A1 receptor agonist N⁶-cyclopentyladenosine (N6-CPA, purity > 98%) was obtained from Abcam (Shanghai, China) and dissolved in DMSO at 50 mM. The adenosine A1 receptor antagonist rolofylline (KW-3902, purity > 99%) was obtained from Tocris Bioscience (Bristol, United Kingdom) and dissolved in DMSO at 10 mM. The adenosine A2a receptor agonist CGS 21680 (purity > 98%) was obtained from Abcam and dissolved in DMSO at 10 mM. The adenosine A2a receptor antagonist SCH 58261 (purity > 99%) was obtained from Abcam and dissolved in DMSO at 10 mM. The adenosine A2b receptor agonist BAY 60-6583 (purity > 98%) was obtained from Tocris Bioscience and dissolved in DMSO at 20 mM. The adenosine A2b receptor antagonist PSB603 (purity > 98%) was obtained from Tocris Bioscience and dissolved in DMSO at 10 mM. The adenosine A3 receptor agonist piclidenoson (IB-MECA, purity > 97%) was obtained from Abcam and dissolved in DMSO at 10 mM. The adenosine A3 receptor antagonist MRE 3008F20 (purity > 98%) was obtained from Tocris Bioscience and dissolved in DMSO at 50 mM. The adenosine analog 5′-N-ethylcarboxamidoadenosine (NECA, purity > 99%) was obtained from Tocris Bioscience and dissolved in DMSO at 50 mM. All solutions were stored at –20°C and used within 1 year.

Tang J., Zou Y., Li L., Lu F., Xu H., Ren P., Bai F., Niedermann G, & Zhu X. (2021). BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor. Frontiers in Pharmacology, 12, 619800.

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9

Isolation and Culture of Murine Intestinal Crypts

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Proximal small intestines of Villin^Cre mice were flushed with ice-cold PBS. Tissue was incubated in 10mL of crypt isolation buffer (1xPBS-2mM EDTA) on a rocker for 5 min at 4°C. Tissue was cut longitudinally into small fragments roughly 0.8cm long and incubated in fresh isolation buffer on a rocker for 45 min at 4°C. After incubation, tubes were shaken vigorously until tissue floated, indicating removal of the mucosal layer. Cell suspensions were passed through 70μm cell strainer. Cells were washed with 1 × DMEM and then centrifuged at 50 × g for 10 min. Crypts were counted by hemocytometer and plated at 350 crypts per well in a 1:1 ratio of Matrigel™ Membrane Matrix Growth Factor Reduced (Corning, Cat #354230) to Advanced DMEM/F-12 (Thermo Scientific, catalog #12634-010). Enteroids were cultured for five days in IntestiCult™ Organoid Growth Medium (StemCell Technologies, Vancouver, Canada, catalog # 06005). Wells were treated with 10 μm of BAY 60–6583 (Tocris Bioscience, Bristol, UK, catalog# 4472) or vehicle control (DMSO) for 48 h. Organoids were harvested and lysed in RIPA buffer with 2× protease inhibitors and 2× EDTA (Thermo Scientific, catalog# 78444). Cell lysate protein concentration was determined using Bradford protein assay kit as explained above.

El-Naccache D.W., Chen F., Palma M.J., Lemenze A., Fischer M.A., Wu W., Mishra P.K., Eltzschig H.K., Robson S.C., Di Virgilio F., Yap G.S., Edelblum K.L., Haskó G, & Gause W.C. (2022). Adenosine metabolized from extracellular ATP promotes type 2 immunity through triggering A2BAR signaling in intestinal epithelial cells. Cell reports, 40(5), 111150.

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10

Isolation and Culture of Murine Intestinal Crypts

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Proximal small intestines of Villin^Cre mice were flushed with ice-cold PBS. Tissue was incubated in 10mL of crypt isolation buffer (1xPBS-2mM EDTA) on a rocker for 5 min at 4°C. Tissue was cut longitudinally into small fragments roughly 0.8cm long and incubated in fresh isolation buffer on a rocker for 45 min at 4°C. After incubation, tubes were shaken vigorously until tissue floated, indicating removal of the mucosal layer. Cell suspensions were passed through 70μm cell strainer. Cells were washed with 1 × DMEM and then centrifuged at 50 × g for 10 min. Crypts were counted by hemocytometer and plated at 350 crypts per well in a 1:1 ratio of Matrigel™ Membrane Matrix Growth Factor Reduced (Corning, Cat #354230) to Advanced DMEM/F-12 (Thermo Scientific, catalog #12634-010). Enteroids were cultured for five days in IntestiCult™ Organoid Growth Medium (StemCell Technologies, Vancouver, Canada, catalog # 06005). Wells were treated with 10 μm of BAY 60–6583 (Tocris Bioscience, Bristol, UK, catalog# 4472) or vehicle control (DMSO) for 48 h. Organoids were harvested and lysed in RIPA buffer with 2× protease inhibitors and 2× EDTA (Thermo Scientific, catalog# 78444). Cell lysate protein concentration was determined using Bradford protein assay kit as explained above.

El-Naccache D.W., Chen F., Palma M.J., Lemenze A., Fischer M.A., Wu W., Mishra P.K., Eltzschig H.K., Robson S.C., Di Virgilio F., Yap G.S., Edelblum K.L., Haskó G, & Gause W.C. (2022). Adenosine metabolized from extracellular ATP promotes type 2 immunity through triggering A2BAR signaling in intestinal epithelial cells. Cell reports, 40(5), 111150.

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Bay 60 6583

Lab products found in correlation

31 protocols using bay 60 6583

Isolation and Purification of Kupffer Cells

Binding Assays for Adenosine Receptors

Nebulized Adora2b Agonist for ALI

In Vivo Administration of BAY 60-6583

In Vitro Osteoclastogenesis Assay

Sprague Dawley Rat Biochemical Assay

Adora2b Deficiency and DSS-Induced Colitis

Adenosine Receptor Ligands and Analogs

Isolation and Culture of Murine Intestinal Crypts

Isolation and Culture of Murine Intestinal Crypts

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