All reagents were commercially available and purchased from various manufacturers. Radioligands [3H]DPCPX (120 Ci/mmol) and [3H]NECA (27.1 Ci/mmol) were obtained from PerkinElmer. Adenosine deaminase from bovine spleen (157 units/mg, 5.9 mg/ml, or 130 units/mg, 6.8 mg/ml), CPA, DPCPX, istradefylline, caffeine and anhydrous magnesium chloride (MgCl2) were all obtained from Sigma-Aldrich. Radioactivity was counted by a PerkinElmer Tri-CARB 2810 TR liquid scintillation analyser.
Istradefylline
Manufactured by Merck Group
Sourced in Poland, United States, United Kingdom
Istradefylline is a laboratory equipment product manufactured by Merck Group. It is an adenosine A2A receptor antagonist. The core function of Istradefylline is to inhibit the activity of the adenosine A2A receptor, which is involved in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Dpcpx, by Merck Group (3 mentions)
Bay 60 6583, by Bio-Techne (2 mentions)
3h dpcpx, by PerkinElmer (1 mentions)
3h neca, by PerkinElmer (1 mentions)
Caffeine, by Merck Group (1 mentions)
Methylcellulose, by Fujifilm (1 mentions)
Rolipram, by Fujifilm (1 mentions)
Theophylline, by Merck Group (1 mentions)
Icr mice, by Charles River Laboratories (1 mentions)
Mrs1754, by Bio-Techne (1 mentions)
Psb1115, by Santa Cruz Biotechnology (1 mentions)
Vetorphale, by Meiji Seika Pharma (1 mentions)
Cgs21680, by Abcam (1 mentions)
Domitor, by Meiji Seika Pharma (1 mentions)
Kw6002, by Merck Group (1 mentions)
Imipramine hydrochloride, by Merck Group (1 mentions)
Adenosine, by Merck Group (1 mentions)
Adenosine, by Bio-Techne (1 mentions)
Adenosine triphosphate (atp), by GE Healthcare (1 mentions)
2 chloro n6 cyclopentyladenosine ccpa, by Bio-Techne (1 mentions)
7 protocols using istradefylline
1
Radioligand Binding Assay for Adenosine Receptors
2
Evaluation of Adenosine Receptor Modulators
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PSB1115 was purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Methylcellulose, ibudilast and rolipram were obtained from Wako Pure Chemical Industries (Osaka, Japan). Medetomidine chloride (Domitor®) and butorphanol tartrate (Vetorphale®) were obtained from Meiji Seika Pharma Co., Ltd. (Tokyo, Japan) and midazolam was purchased from SANDOZ (Tokyo, Japan). Istradefylline and theophylline were purchased from Sigma (St. Louis, MO). Cilostazol and aminophylline were obtained from LKT laboratories, Inc. (St Paul, MN). MRS-1754 and BAY60-6583 were from Tocris Bioscience (Bristol, UK) and 1,3-dipropyl-8-cyclopentylxanthine (DCPCX), ZM241385 and CGS21680 were from Abcam (Cambridge, UK). ICR mice (5- or 6-week-old males, 28–33 g), primiparous late pregnant Wistar female rats and normal male Wistar rats (4- or 5-week-old, 150–250 g) were obtained from Charles River Laboratories Japan (Yokohama, Japan). The animals were housed under conditions of controlled temperature (22–24 °C) and illumination (12-h light cycle) conditions for 1 or 2 weeks before experiments. The experiments and procedures described here were performed in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health, and were approved by the Animal Care Committees of Keio University and St. Marianna University.
3
Targeted Phrenic Neuron Depletion and Rescue
4
Synergistic Effects of Adenosine Antagonists
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The following substances were used: DPCPX (8–cyclopentyl–1,3–dipropylxanthine, Sigma–Aldrich, Poznań, Poland), istradefylline (KW–6002, (E)–8–(3,4–dimethoxystyryl)– 1,3–diethyl–7–methylxanthine, Sigma–Aldrich, Poznań, Poland), magnesium hydroaspartate (Farmapol, Poznań, Poland), and zinc hydroaspartate (Farmapol, Poznań, Poland). DPCPX (1 mg/kg) and istradefylline (0.5 mg/kg) were suspended in a 0.9% saline with Tween 80 (1%) (POCH, Gliwice, Poland), whereas magnesium and zinc hydroaspartate (10 mg/kg and 2.5 mg/kg, calculated as pure magnesium or zinc ions, respectively) were dissolved in 0.9% saline. DPCPX and magnesium hydroaspartate were injected intraperitoneally (i.p.) 30 min, while istradefylline was administered orally (p.o.) and zinc hydroaspartate i.p. 60 min before behavioural testing. Animals from control group were given 0.9% saline. All liquid dosage forms were prepared immediately prior to the experiments and they were administered in a volume of 0.01 mL/g.
The treatment schedules and subtherapeutic doses of DPCPX, Mg2+ and Zn2+ were chosen on the basis of our previous projects [30 (link),31 (link)], whereas of istradefylline were selected on the basis of literature data [105 (link)] and then confirmed in preliminary studies carried out in our laboratory.
The treatment schedules and subtherapeutic doses of DPCPX, Mg2+ and Zn2+ were chosen on the basis of our previous projects [30 (link),31 (link)], whereas of istradefylline were selected on the basis of literature data [105 (link)] and then confirmed in preliminary studies carried out in our laboratory.
5
Age-related Istradefylline Treatment
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Drug solution was made by dissolving istradefylline (Sigma Aldrich, St. Louis, MO, USA) in 2% DMSO and 0.9% saline, then, aliquoted and stored at −20 °C. istradefylline was injected to wild-type C57BL/6J mice intraperitoneally once a week at a dose of 1 mg/kg, commencing at the age of 6 months until the age of 12 months. An equivalent volume of the drug vehicle solution was administered by intraperitoneal injection to the control group of mice once a week.
6
Pharmacological Evaluation of Selenium-Mediated Effects
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Sodium selenite (Se), DPCPX (8-cyclopentyl-1,3-dipropylxanthine), istradefylline (IST, (E)-8-(3,4-dimethoxystyryl)-1,3-diethyl-7-methylxanthine), and imipramine hydrochloride (IMI) were purchased from Sigma-Aldrich (Poznań, Polska). Se was dissolved in 0.9% NaCl and administered intraperitioneally (i.p.) 30 min prior behavioral testing at doses of 0.125, 0.25, 0.5, and 1.0 mg/kg, which refers to 0.056, 0.1125, 0.225, and 0.45 mg/kg of pure selenium, respectively. DPCPX and IST were suspended in 1% solution of Tween 80 (POCH, Gliwice, Polska) in saline (0.9% NaCl). DPCPX was injected i.p at a dose of 1 mg/kg, while IST was administered per os (p.o.) at a dose of 0.5 mg/kg 60 min before the experiment. IMI (30 mg/kg), used as a reference drug (a positive control group) in Se dose-effect studies, was dissolved in 0.9% NaCl and injected i.p. 60 min before behavioral studies. The control group received 0.9% NaCl at the respective time prior to the experiment. All solutions/suspensions were prepared immediately before behavioral tests and administered in a volume of 0.01 mL/g body weight. Each experimental group consisted of 10 mice, randomly assigned prior to administration of a tested substance.
7
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.
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