Mibefradil

Manufactured by Bio-Techne

Sourced in United Kingdom

Mibefradil is a lab equipment product offered by Bio-Techne. It functions as a calcium channel blocker, specifically targeting T-type calcium channels. This product is intended for research use only, and its core function is to facilitate the study of calcium channel dynamics in various experimental settings.

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Lab products found in correlation

13 protocols using mibefradil

Obtaining Mibefradil, Azamulin, and Bergamottin

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Mibefradil was obtained from Tocris Bioscience (Minneapolis, MN, USA) azamulin and DHB from Cayman Chemical (Ann Arbor, MI, USA), and bergamottin from Sigma-Aldrich (St. Louis, MO, USA).

, & Sevrioukova I.F. (2019). Structural Insights into the Interaction of Cytochrome P450 3A4 with Suicide Substrates: Mibefradil, Azamulin and 6′,7′-Dihydroxybergamottin. International Journal of Molecular Sciences, 20(17), 4245.

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Isolation of Neuronal Ca2+ Currents

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Neurons were isolated from their surrounding synaptic environment by blocking AMPA, NMDA and GABA_A and glycine receptors with CNQX (20 μM, Tocris), AP5 (50 μM, Tocris), Gabazine (10 μM, Tocris) and Strychnine (10 μM, Sigma-Aldrich). Ca²⁺ currents were isolated by blocking voltage gated K⁺ channels with 4-Aminopyridine (4-AP, 3 mM; Sigma-Aldrich), tetraethylammonium (TEA, 0.1 mM; Sigma-Aldrich) cesium (1 mM; Sigma-Aldrich) and Na⁺ channels with tetrodotoxin (TTX, 1 μM; Alomone Labs). Drugs applied to the extracellular medium: 5-HT hydrochloride (10 μM; Sigma Aldrich); 4-Iodo-2,5-dimethoxy-α-methylbenzeneethanamine hydrochloride (DOI hydrochloride; 10–20 μM, Tocris); Mibefradil (8–16 μM, Tocris).

Petersen A.V., Jensen C.S., Crépel V., Falkerslev M, & Perrier J.F. (2017). Serotonin Regulates the Firing of Principal Cells of the Subiculum by Inhibiting a T-type Ca2+ Current. Frontiers in Cellular Neuroscience, 11, 60.

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Striatal Slice Preparation and Neuromodulation

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Mice were anesthetized using isofluorane and then decapitated. Brains were removed, cut into left and right hemispheres, and then 300 μm coronal slices were made at 1-4°C in oxygenated (95% v/v O₂, 5% v/v CO₂) dissecting solution (208 mM sucrose, 2.5 mM KCl, 1 mM CaCl₂, 4 mM MgCl₂, 4 mM MgSO₄, 1.6 mM NaH₂PO₄, 26 mM NaHCO₃, 10 mM glucose, and 3 mM Na-pyruvate) using a Vibratome 3000 (The Vibratome Company). Typically, a total of 5-7 striatal hemi-slices (left/right hemisphere combined) were obtained from each mouse brain (1.1 to 0.14 mm from Bregma). Slices were allowed to recover on a nylon mesh for 1 h at 30°C in oxygenated ACSF (113 mM NaCl, 2.5-5 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1 mM NaH₂PO₄, 26 mM NaHCO₃, 20 mM glucose, and 3 mM Na-pyruvate) followed by addition of picrotoxin (50 μM) for 30 min. Slices were then transferred to oxygenated 30°C ACSF solutions supplemented with vehicle or drug for 1-30 min, as defined in the figure legends. The following drugs were used and dissolved in water or DMSO based on manufacturer's instructions: BAPTA (Sigma), (S)-BayK8644 (Tocris), FPL64176 (Tocris), isradipine (National Institute of Mental Health Chemical Synthesis and Drug Supply Program), KCl (Sigma), mibefradil (Tocris), Nickel Chloride (Sigma), nimodipine (MP Biomedicals), SNX-482 (Peptides International), and TTX (Tocris).

Pasek J.G., Wang X, & Colbran R.J. (2015). Differential CaMKII regulation by voltage-gated calcium channels in the striatum. Molecular and cellular neurosciences, 68, 234-243.

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Pharmacological Modulation of Neural Signaling

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Picrotoxin, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), trans-2-Carboxy-5,7-dichloro-4-phenylaminocarbonylamino-1,2,3,4-tetrahydro-quinoline (L-689560), Nimodipine, Mibefradil, apamin, TTX, DHPG, MPEP and 6-amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM298198) hydrochloride were purchased from Tocris. Fluo-5F and Alexa Fluor 594 were purchased from Invitrogen. All other chemicals were purchased from Fisher Scientific.

Tigaret C.M., Olivo V., Sadowski J.H., Ashby M.C, & Mellor J.R. (2016). Coordinated activation of distinct Ca2+ sources and metabotropic glutamate receptors encodes Hebbian synaptic plasticity. Nature Communications, 7, 10289.

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Odorant Detection Signaling Pathways

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All odorants, including Troenan, were purchased from Sigma Aldrich (Munich, Germany) and Henkel (Düsseldorf, Germany). The inhibitor L-cis-diltiazem was purchased from Abcam (Cambridge, MA, USA), SQ22.536, Thapsigargin and U-73122 were purchased from Sigma Aldrich, and the TRP-channel inhibitor Ruthenium red was purchased from Abcam. BTP-2, Mibefradil and Gallein were purchased from Tocris (Bristol, UK). Ringer’s solution used in the calcium imaging experiments contained 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂ and 10 mM HEPES, pH 7.4. Forskolin used in the CRE-Luciferase assay was obtained from Sigma Aldrich. Like all tested odorants and inhibitors, it was prediluted in DMSO prior to usage.

Weber L., Al-Refae K., Ebbert J., Jägers P., Altmüller J., Becker C., Hahn S., Gisselmann G, & Hatt H. (2017). Activation of odorant receptor in colorectal cancer cells leads to inhibition of cell proliferation and apoptosis. PLoS ONE, 12(3), e0172491.

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Culturing HAT-7 Cells with Activators

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HAT-7 cells [21 (link)] were grown in Dulbecco’s modified Eagle’s medium and Nutrient Mixture F-12 Ham medium (DMEM:F12; Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% HyClone characterised fetal bovine serum (FBS; Cytiva, Marlborough, MA, USA), 100 U/mL penicillin and 100 mg/mL streptomycin (Sigma Aldrich, St. Louis, MO, USA). Cells were maintained at 37 °C in 5% CO₂ and passaged every 4 days with 0.25% trypsin-EDTA (Thermo Scientific, Waltham, MA, USA). Following activators and inhibitors were used in functional studies: naltriben (Tocris Bioscience, Bristol, UK), mibefradil (Tocris Bioscience, Bristol, UK), NS8593 (Tocris Bioscience, Bristol, UK), thapsigargin (Sigma Aldrich, St. Louis, MO, USA), FTY720 (Sigma Aldrich, St. Louis, MO, USA) and BTP2 (Merck KGaA, Dramstadt, Germany).

Kádár K., Juhász V., Földes A., Rácz R., Zhang Y., Löchli H., Kató E., Köles L., Steward M.C., DenBesten P., Varga G, & Zsembery Á. (2021). TRPM7-Mediated Calcium Transport in HAT-7 Ameloblasts. International Journal of Molecular Sciences, 22(8), 3992.

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Intravitreal Injections for RGC Analysis

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The procedure for intravitreal injections was described in our previous studies (Ji et al., 2012; Dong et al., 2015). After the pupil of the anesthetized eye was dilated with tropicamide drops (Santen Pharmaceutical, Shanghai, China), a single dose of TNF-α (5 ng/mL, R&D Systems, Minneapolis, MN, USA) or XPro1595 (50 µg/mL; Xencor, Monrovia, CA, USA) + TNF-α (5 ng/mL) in 0.9% saline (2 µL) was intravitreally injected 3 days before the RGCs were isolated for Ca²⁺ current recordings and western blotting using a micro-injector with a 30-gauge needle (Hamilton, Reno, NV, USA) under a stereoscopic microscope (Carl Zeiss, Oberkochen, Germany). For Ca²⁺ current recordings in RGCs isolated from COH retinas, a single dose of XPro1595 (50 µg/mL) dispersed in 0.9% saline (2 µL) was intravitreally injected 1 day before the COH operation. For terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) staining, a single dose of mibefradil (3 µM; Tocris Bioscience) in 0.9% saline (2 µL) was intravitreally injected 1 day before the COH operation. In vehicle (sham) controls, eyes were injected with saline in the same manner.

Wang H.N., Qian W.J., Zhao G.L., Li F., Miao Y.Y., Lei B., Sun X.H, & Wang Z.F. (2022). L- and T-type Ca2+ channels dichotomously contribute to retinal ganglion cell injury in experimental glaucoma. Neural Regeneration Research, 18(7), 1570-1577.

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Electrophysiological Recordings with Pharmacological Agents

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Nimodipine, mibefradil, PDTC (Tocris Bioscience), and U0126 (Tocris Bioscience) were dissolved in dimethyl sulfoxide and added to the bath solution immediately before electrophysiological recordings. The final concentration of dimethyl sulfoxide in the solution was equal to or less than 0.1%; this concentration has been demonstrated to have no significant effects on Ca²⁺ currents in RGCs (Qian et al., 2017). The other chemicals were freshly dissolved in the bath solution. The drugs were delivered through a stepper motor-based rapid solution changer (RSC-160, Bio-Logic, Claix, France) (Zhao et al., 2010; Li et al., 2016; Qian et al., 2017).

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Evaluation of Ion Channel Modulators

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NS8593 (N2538), NiCl₂.6H₂O (N6136) and MgCl₂.6H₂O (M2393) were purchased from Sigma Aldrich. Mibefradil (2198) was from Tocris. 2-APB (100065), Thapsigargin (586005) and Ionomycin (407592) were from Calbiochem. Waixenicin A was provided as a generous gift from Dr. F. David Horgen (Hawaii Pacific University; Kaneohe, HI). Stock solutions were made following manufacturers’ recommendations and kept in −80 °C.

Ardestani G., Mehregan A., Fleig A., Horgen F.D., Carvacho I, & Fissore R.A. (2020). Divalent Cation Influx and Calcium Homeostasis in Germinal Vesicle Mouse Oocytes. Cell calcium, 87, 102181.

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Pharmacological Modulation of Neural Activity

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For pharmacological experiments, drug effects were recorded at 5–10 minutes after the addition of the compounds to the perfusion fluid. Agents were used at the following concentrations: mibefradil (Tocris): 20 μM, 2-amino-5-phosphonopentanoic acid (AP5, Tocris): 50 μM, tetrodotoxin (TTX, Tocris): 100 nM, picrotoxin (PTX, Tocris): 6 μM, NBQX (Tocris): 10 μM, 4-AP (Tocris): 100–500 μM. For cell autonomous inhibition of Na⁺ channels, QX 314 (HelloBio) was added to the intracellular solution in 5 mM concentration. To specifically block postsynaptic GABA_A receptors, we included picrotoxin in the pipette solution at 10 μM concentration (Inomata et al., 1988 (link); Metherate and Ashe, 1993 (link); Yazaki-Sugiyama et al., 2009 (link)). All other materials, including standard salts, were acquired from Sigma or Fisher Scientific.

Rindner D.J., Proddutur A, & Lur G. (2022). Cell-type specific integration of feedforward and feedback synaptic inputs in the posterior parietal cortex. Neuron, 110(22), 3760-3773.e5.

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