COS-7 cells expressing wild-type or mutant alleles of ATP1A3 were assessed in the ATPase assay as described previously (4 (link)). Briefly, synthesized adenosine 5′-diphosphate (ADP) by ATPase reaction was detected using the EnzyLight ADP Assay Kit (BioAssay Systems, Hayward, CA, USA). ATPase activity was estimated by subtracting the luminescence signal after a 1-hour pretreatment with 100 μM ouabain (Sigma-Aldrich, St. Louis, MO, USA) from the luminescence reading without ouabain.
Ouabain
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Sao Tome and Principe, Macao, India, France, Denmark, Poland
Ouabain is a cardiac glycoside that is used as a reference standard and research tool in the study of cardiac function and ion transport mechanisms. It acts by inhibiting the Na+/K+ ATPase pump, which is essential for maintaining the electrochemical gradient across the cell membrane. Ouabain is commonly used in various in vitro and in vivo experiments, particularly in the fields of physiology, pharmacology, and biochemistry.
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Lab products found in correlation
Digoxin, by Merck Group (14 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (9 mentions)
Amiloride, by Merck Group (9 mentions)
Bumetanide, by Merck Group (8 mentions)
Nifedipine, by Merck Group (7 mentions)
Bovine serum albumin (bsa), by Merck Group (7 mentions)
Indomethacin, by Merck Group (6 mentions)
Kb r7943, by Merck Group (6 mentions)
Potassium chloride (kcl), by Merck Group (5 mentions)
Adenosine triphosphate (atp), by Merck Group (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Lipopolysaccharides (lps), by Merck Group (4 mentions)
Digitonin, by Merck Group (4 mentions)
Ampicillin, by Merck Group (4 mentions)
Rotenone, by Merck Group (4 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (4 mentions)
L name, by Merck Group (4 mentions)
Latrunculin a, by Merck Group (4 mentions)
Digitoxin, by Merck Group (4 mentions)
Digitoxigenin, by Merck Group (4 mentions)
193 protocols using ouabain
1
ATPase activity assay of ATP1A3
2
Inhibiting Na+,K+-ATPase and Actin Dynamics in Zebrafish Embryos
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In order to inhibit the activity of Na+,K+-ATPase, embryos at the 20 hpf stage were soaked in 1% DMSO + ouabain (Sigma Aldrich, CAS 11018-89-6) across a range of concentrations from 0 to 1 mM. ouabain was stored at 10 mM in 1% DMSO and diluted to required concentrations in 1X Danieau buffer before use. Ear size was assessed at 30 hpf as an endpoint. For long-term imaging, ouabain was added to 1% agarose mold used for mounting the embryos. To ensure consistent penetration, 2.3 nl of 0.75 mM ouabain was injected (Nanoject) into the cardiac chamber for circulation throughout the embryo. Assuming an average embryo volume of ∼180 nl, this injected dose guarantees an effective concentration of 10 µM. In order to perturb the actin network in the otic vesicle, 2.3 nl of 2 mM cytochalasin D was injected into the cardiac chamber for an effective concentration of 25 µM in the ear.
3
Modulating Intracellular Sodium Levels
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To increase the rate-limiting intracellular Na+ concentration oocytes were incubated in Na+-loading solution (in mM, 90 NaOH, 20 TEA-OH, 40 HEPES, and 0.2 EGTA) with 10 µM ouabain one hour before the two-electrode voltage clamp (TEVC) experiments. Such ouabain concentration inhibited endogenous pumps for the duration of the experiments leaving exogenous pumps unaffected [19 (link)].
TEVC was performed with an OC-725C amplifier (Warner Instruments) controlled by pClamp software and a Digidata 1440A (Molecular Devices). Current signals were acquired at 10 kHz and also continuously recorded at 1 kHz via a Minidigi 1B (Molecular Devices). The current and voltage glass microelectrodes were filled with 3 M KCl and had resistances of 0.2–1 MΩ. Oocytes were clamped at a holding potential of − 50 mV from which square voltage steps were applied as indicated.
Experiments were carried out at room temperature 19–21 °C in an extracellular solution containing 122 mM methanesulfonic acid (MS), 5 mM Ba(OH)2, 1 mM Mg(OH)2, 0.5 Ca(OH)2, 10 mM HEPES, 125 mM NaOH (titrated to pH 7.6 with MS, osmolality of 250–260 mOsm/kg). K+ was added to the external solution from a 450 mM K-MS stock. ouabain (Sigma-Aldrich) was directly dissolved at 10 mM in the external solutions to reversibly inhibit the ouabain-resistant exogenously expressed pumps.
TEVC was performed with an OC-725C amplifier (Warner Instruments) controlled by pClamp software and a Digidata 1440A (Molecular Devices). Current signals were acquired at 10 kHz and also continuously recorded at 1 kHz via a Minidigi 1B (Molecular Devices). The current and voltage glass microelectrodes were filled with 3 M KCl and had resistances of 0.2–1 MΩ. Oocytes were clamped at a holding potential of − 50 mV from which square voltage steps were applied as indicated.
Experiments were carried out at room temperature 19–21 °C in an extracellular solution containing 122 mM methanesulfonic acid (MS), 5 mM Ba(OH)2, 1 mM Mg(OH)2, 0.5 Ca(OH)2, 10 mM HEPES, 125 mM NaOH (titrated to pH 7.6 with MS, osmolality of 250–260 mOsm/kg). K+ was added to the external solution from a 450 mM K-MS stock. ouabain (Sigma-Aldrich) was directly dissolved at 10 mM in the external solutions to reversibly inhibit the ouabain-resistant exogenously expressed pumps.
4
Ouabain-Induced Ventricle Injection
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ICV injections were performed by inserting injection cannulas into the guide cannulas 2.5 mm deep following a 2-day rehabilitation period after surgery. Injection cannulas were connected to Hamilton syringes by polyethylene tubing (i.d. 0.28 mm). 0.5 μl of artificial cerebrospinal fluid (aCSF: 125 mM NaCl, 26 mM NaHCO3, 4 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl2, 2 mM MgCl2, 25 mM glucose) or 50 µM ouabain (Sigma, USA) in aCSF was injected into the left and right ventricles for 2 min at a speed of 0.5 μl/min using a syringe pump (0.5 μl in each ventricle). After injections, mice were immediately tested in the open field. In the haloperidol and haloperidol + ouabain-treated groups of animals 5 µg/ml haloperidol (Sigma, USA) in 0.9% NaCl (intraperitoneally, 70 µg/kg) was administered 30 min before the ICV injections.
5
Electrophysiological Muscle Preparation
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Control solution contained (mM): 118.5 NaCl, 4.7 KCl, 1.3 CaCl2, 3.1 MgCl2, 25 NaHCO3, 2 NaH2PO4, and 5.5 d -glucose. Solutions containing different K+ concentrations were prepared by adding the appropriate amount of KCl. Solutions containing ouabain, an NKA inhibitor (Sigma-Aldrich), were prepared by dissolving ouabain directly in the control solution. Solutions containing 2-[(3,4-dihydro-2-phenyl-2H-1-benzopyran-6-yl)oxy]-5-nitro-pyridine (ORM-10103; an inhibitor of the NCX; Sigma-Aldrich) were prepared by first dissolving ORM-10103 in DMSO before it was added to the control solution. For the experiments involving ORM-10103, the final DMSO concentration was 0.1% (vol/vol) including the control solution. Solutions were continuously bubbled with 95% O2–5% CO2 to maintain a pH of 7.4. Experimental temperature was 37°C. Total flow of solutions in the muscle chamber was 15 ml/min being split just above and below the muscle to prevent any buildup of reactive oxygen species, which is quite large at 37°C (Edwards et al., 2007 (link)).
6
Ouabain, LPS, and Bufagenin Signaling
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7
Ouabain and Nicotine Binding Assay
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Bodipy-conjugated Ouabain was from Invitrogen. Ouabain, nicotine ((−)nicotine hydrogen tartrate) and other chemicals were from Sigma-Aldrich.
Data are given as the mean ± SEM. Statistical significance of the difference between means was evaluated using a Student's t-test (ORIGIN 6.1. software) and one way ANOVA (ORIGIN Pro 8 software).
Data are given as the mean ± SEM. Statistical significance of the difference between means was evaluated using a Student's t-test (ORIGIN 6.1. software) and one way ANOVA (ORIGIN Pro 8 software).
8
Mitochondrial Dysfunction Evaluation Protocol
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Apocynin, apyrase, bovin serum albumin, butylated hydroxytoluene, cytochrome C, dichlorofluorescin diacetate (DCFH-DA), diphenyleneiodonium (DPI), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), dithiotreitol (DTT), L-lactic dehydrogenase (EC 1.1.1.27), leupeptin, NAD+, NADH, PGE1, piceatannol, phenylmethylsulfonyl fluoride (PMSF), PP2 analogue (PP2), protease inhibitor cocktail (Cat. N° P8340), superoxide dismutase (SOD), thiobarbituric acid (TBA), digitonin, pyruvate, malate, succinate, ouabain, ampicillin, di-adenosine-5'penta-phosPHAte, rotenone, antimycin A, ADP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) and all chemicals were from Sigma-Aldrich, USA. Lectins (WGA, PHA, LCA) and LY294002 were purchased from Merck Millipore, Germany. 2(trifluoromethyl)phenothiazine (2TFP), specific inhibitor of Nox1 [30 (link)], was a gift of Prof. Bruno Tasso Dept. PHArmacy, Genoa University, Genoa. Apocynin, DPI, DTT, LY294002, piceatannol, PP2, 2TFP and FCCP, were diluted in saline from a stock DMSO solution immediately before each experiment. MitoProbe™ Tetramethylrhodamine Methyl Ester (TMRM) from ThermoFisher Scientific was a gift from Dr. Paolo Degan, UO Mutagenesi e Prevenzione Oncologica, IRCCS Ospedale Policlinico San Martino, Genoa. ATP bioluminescence assay kit CLSII and ATP standard solution were from Roche, Switzerland.
9
Automated Patch Clamp Study of Neuroblastoma Cells
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Tetracaine hydrochloride and lidocaine hydrochloride monohydrate were obtained from Merck KGaA (Darmstadt, Germany). Tetrodotoxin standard was purchased from Tocris Bioscience (Bristol, UK) and the standard solution was prepared at 1 mg/mL in 1% acetic acid. The automated patch clamp Patchliner with 8 amplifier channels, 2 HEKA EPC10 Quadro amplifiers, NPC-16 borosilicate recording chips (medium resistance), external solution (140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM D-glucose monohydrate, 10 mM HEPES/NaOH, pH 7.4), internal solution (50 mM CsCl, 10 mM NaCl, 60 mM CsF, 20 mM EGTA, 10 mM HEPES/CsOH, pH 7.2), and seal enhancer solution (10 mM HEPES, 130 mM NaCl, 5 mM glucose, 4 mM KCl, 10 mM CaCl2, 1 mM MgCl2, pH 7.4, MOsm 302) were obtained from Nanion Technologies GmbH (Munich, Germany). Neuroblastoma murine (Neuro-2a) cells were purchased from ATCC LGC standards (Manassas, VA, USA). Foetal bovine serum (FBS), penicillin/streptomycin solution, phosphate-buffered saline (PBS), Roswell Park Memorial Institute (RPMI) medium, sodium pyruvate, trypsin-EDTA enzyme, ouabain, veratridine, and thiazolyl blue tetrazolium bromide (MTT) were purchased from Merck KGaA (Darmstadt, Germany).
10
Quantification of Lipid Peroxidation Markers
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The materials used in this research are as follows: 1,3,3-tetraethoxypropane (Sigma, CAS number 122-31-6), 2-thiobarbituric acid (Merck, CAS number 504-17-6), 5,5′-dithiobis(2-nitrobenzoic acid) (Sigma, CAS number 69-78-3), acetic acid (Merck, CAS number 64-19-7), acetonitrile (Merck, CAS number 75-05-8), EGTA (Sigma, CAS number 67-42-5), FeCl3 (Sigma, CAS number 7705-08-0), FeSO4 (Sigma, CAS number 7720-78-7), KCl (Merck, CAS number 7447-40-7), methanol (Merck, CAS number 67-56-1), MgCl2 (Merck, CAS number 7786-30-3), Na2EDTA (Merck, CAS number 6381-92-6), NaCl (Sigma, CAS number 7647-14-5), ouabain (Merck, CAS number 11018-89-6), sodium citrate (Merck, CAS number 6132-04-3), sodium dodecyl sulfate (Merck, CAS number 151-21-3), tetrahydrofuran (Merck, CAS number 109-99-9), TPTZ (Sigma, CAS number 3682-35-7), trichloroacetic acid (Merck, CAS number 76-03-9), and Tris hydrochloride (Sigma, CAS number 1185-53-1).
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