Murine CFs were isolated from healthy A2bR–/– mice or their littermate controls by collagenase digestion using the Langendorff methods and magnetic bead–mediated enrichment as described above. Isolated CFs were cultivated in DMEM complete (4,500 mg/L glucose, 20% FCS, 1% penicillin and streptomycin, 1% GlutaMAX). All experiments were conducted with cells in passage 2: 60,000 CFs were seeded per well of a 12-well plate and permitted to settle overnight (passage 2). Cells were washed with DMEM complete and incubated with 400 μL DMEM complete containing 33 μM erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) (an adenosine deaminase [ADA] inhibitor, Tocris) and 33 μM nitrobenzylthioinosine (NBMPR) (an inhibitor of equilibrative nucleoside transporter 1 ENT1, Tocris), with or without 1 μM FR900359 (a Gq inhibitor, provided by Evi Kostenic, University of Bonn, Bonn, Germany) for 10 minutes at 37°C. Afterwards, 100 μL DMEM complete was added and contained (besides 33 μM EHNA and 33 μM NBMPR with or without 1 μM FR900359) 100 μM adenosine (Tocris) or only the corresponding amount of DMSO (vehicle control), giving a final concentration of 20 μM adenosine. After incubation for 24 hours, supernatant was collected and used for cytokine analysis with Bioplex technology (Bio-Rad) or IL-11 ELISA (Thermo Fisher Scientific). Cells were washed with PBS and used for mRNA expression analysis.
Adenosine
Manufactured by Bio-Techne
Sourced in United Kingdom
Adenosine is a nucleoside compound composed of adenine and ribose. It is a key intermediate in cellular energy transfer, as it is a precursor to adenosine triphosphate (ATP), the primary energy currency of cells. Adenosine plays a role in various physiological processes, including vasodilation, modulation of neurotransmitter release, and regulation of sleep-wake cycles.
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Lab products found in correlation
Sch58261, by Bio-Techne (2 mentions)
Cgs21680, by Bio-Techne (2 mentions)
Caffeine, by Bio-Techne (2 mentions)
Psb0777, by Bio-Techne (2 mentions)
Arl67156, by Bio-Techne (2 mentions)
Forskolin, by Bio-Techne (1 mentions)
Sulpiride, by Bio-Techne (1 mentions)
Dopamine, by Merck Group (1 mentions)
Dpcpx, by Bio-Techne (1 mentions)
Sch23390, by Bio-Techne (1 mentions)
Thiostrepton, by Merck Group (1 mentions)
Mevalonolactone, by Merck Group (1 mentions)
Pitavastatin, by Selleck Chemicals (1 mentions)
Simvastatin, by Selleck Chemicals (1 mentions)
Ym 53601, by Cayman Chemical (1 mentions)
Istradefylline, by MedChemExpress (1 mentions)
Nb 598 maleate, by Adooq (1 mentions)
Ggti 298, by Bio-Techne (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Luf 5834, by Bio-Techne (1 mentions)
7 protocols using adenosine
1
Adenosine Signaling in Cardiac Fibroblasts
2
Neurotransmitter Receptor Pharmacology
3
Drug Treatment Protocols and Assays
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Adenosine (Tocris, 3624), FTI-277 (Selleckchem, S7465), GGTI-298 (Tocris, 2430), istradefylline (KW-6002, MedChemExpress HY-10888), NB-598 maleate (AdooQ, A15180–10), simvastatin (Selleckchem, S1792), pitavastatin (Selleckchem, S1759), thapsigargin (Sigma-Aldrich, 586005), thiostrepton (Sigma-Aldrich, 598226), and YM-53601 (Cayman, 18113) were dissolved in DMSO (Thermo Fisher Scientific BP231), and stock solutions were aliquoted and stored at −80°C. FPP (Cayman 63250) and GGPP (Cayman 63330) were supplied as a solution of methanol:NH4OH (70:30) and stored at −20°C. Baclofen (MedChemExpress, HY-B0007) and caffeine (Tocris, 2793) were dissolved in H2O and mevalonolactone (Sigma-Aldrich M4667) was diluted with H2O and stored at −20°C. Cells were treated with individual or combinations of drugs or small molecules at indicated concentrations for various times and collected for trypan blue exclusion assay, qRT-PCR or immunoblotting.
4
Pharmacological Compounds for Cell Assays
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5
Investigating STAT1-IRF1 Signaling Cascades
6
Adenosine Regulation of Splenic Lymphocyte Interactions
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Splenic lymphocytes were collected by lyzing tissue in a Dounce homogenizer, followed by layering over Ficoll Paque (GE health care, Chicago, IL, USA), as described previously (21) .
Balb/c splenic lymphocytes (3 × 10 6 ) were mixed with SJL/J splenic lymphocytes (3 × 10 6 ) in 500 µL of DMEM medium containing 10% FCS, 100 U/ml penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 µM 2-mercaptoethanol (D10 medium) in 24 well plates in the presence of adenosine (0-1 mM) (Sigma, St. Louis, MO, USA); in the presence of each adenosine receptor agonist (0-10 µM) (A1R: 2-Chloro-N6-cyclopentyladenosine (CCPA), Tocris, Bristol, UK; A2aR: PSB0777, Tocris; A2bR: BAY 60-6583, Tocris; A3R: HEMADO, Tocris); in the presence of A2aR antagonist (0-1 nM) (Istradefylline, Sigma) plus adenosine (100 µM); in the presence of an adenyl cyclase inhibitor (0-1 µM) (MDL-12330A, Enzo Life Sciences, Farmingdale, NY, USA) or a protein kinase A inhibitor (0-1 µM) (H-89, Tocris) plus adenosine (100 µM) to inhibit A2aR signaling; or in the presence of a CD39 inhibitor (0-1 µM) (ARL67156, Tocris) or a CD73 inhibitor (0-1 µM) (adenosine 5'-(α, β-methylene) diphosphate (AMP-CP; Tocris) plus ATP (100 µM) (GE healthcare). After mixing, the plates were incubated for 7 days at 37°C. The supernatants were collected for use in cytokine ELISAs.
Balb/c splenic lymphocytes (3 × 10 6 ) were mixed with SJL/J splenic lymphocytes (3 × 10 6 ) in 500 µL of DMEM medium containing 10% FCS, 100 U/ml penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 µM 2-mercaptoethanol (D10 medium) in 24 well plates in the presence of adenosine (0-1 mM) (Sigma, St. Louis, MO, USA); in the presence of each adenosine receptor agonist (0-10 µM) (A1R: 2-Chloro-N6-cyclopentyladenosine (CCPA), Tocris, Bristol, UK; A2aR: PSB0777, Tocris; A2bR: BAY 60-6583, Tocris; A3R: HEMADO, Tocris); in the presence of A2aR antagonist (0-1 nM) (Istradefylline, Sigma) plus adenosine (100 µM); in the presence of an adenyl cyclase inhibitor (0-1 µM) (MDL-12330A, Enzo Life Sciences, Farmingdale, NY, USA) or a protein kinase A inhibitor (0-1 µM) (H-89, Tocris) plus adenosine (100 µM) to inhibit A2aR signaling; or in the presence of a CD39 inhibitor (0-1 µM) (ARL67156, Tocris) or a CD73 inhibitor (0-1 µM) (adenosine 5'-(α, β-methylene) diphosphate (AMP-CP; Tocris) plus ATP (100 µM) (GE healthcare). After mixing, the plates were incubated for 7 days at 37°C. The supernatants were collected for use in cytokine ELISAs.
7
Pharmacological Modulation of Adenosine Signaling
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Compounds (10 μM of AppCH 2 ppA), 10 μM of adenosine, 40 nM of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (Tocris Bioscience, Bristol, UK), 10 μg/mL of adenosine deaminase (ADA) (Merck, Darmstadt, Germany), 100 μM of suramin hexasodium salt, 10 nM of MRS2500 tetraammonium salt (Tocris Bioscience, Bristol, UK), 100 nM of tertiapin-Q (Tocris Bioscience, Bristol, UK), 100 μM of adenosine 5′-(α,β-methylene) diphosphate (ADP-methylene) (Merck, Darmstadt, Germany) and 500 μM of bupivacaine were added to ACSF-2 and superfused with the same rate as control solution. Inosine (Merck, Darmstadt, Germany) at concentration of 100 μM was added to Kglu intracellular solution.
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