Mazindol

Manufactured by Merck Group

Sourced in United States

Mazindol is a lab equipment product manufactured by Merck Group. It is a cyclohexanol derivative that functions as a stimulant. The core function of Mazindol is to act as a norepinephrine-dopamine reuptake inhibitor, which can be used in various research and testing applications.

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Lab products found in correlation

9 protocols using mazindol

Measuring Dopamine Uptake in Neuron-Glia Cultures

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After being stimulated with 150 EU/ml LPS, 250 nM α-Syn aggregates, or 1.0 μM α-Syn peptides, neuron-glia, neuron-astrocyte, and reconstituted neuron-glia cultures were incubated for 20 min at 37 °C with ³H-labeled DA (PerkinElmer Life Sciences, Santa Clara, CA) in Krebs-Ringer buffer (16 mM sodium phosphate, 119 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl₂, 1.2 mM MgSO₄, 1.3 mM EDTA, and 5.6 mM glucose; pH 7.4). Cells were washed with cold Krebs-Ringer buffer and lysed in 1 N NaOH. The radioactivity of the lysates was measured using a liquid scintillation counter (PerkinElmer, Santa Clara, CA) and calculated by subtracting nonspecific DA uptake detected in samples in the presence of 10 μM mazindol (Sigma-Aldrich, St. Louis, MO) from the results of samples that did not include mazindol. Data were presented as the percentage of controls in the same batch of cell cultures [4 (link)].

Wang S., Chu C.H., Guo M., Jiang L., Nie H., Zhang W., Wilson B., Yang L., Stewart T., Hong J.S, & Zhang J. (2016). Identification of a specific α-synuclein peptide (α-Syn 29-40) capable of eliciting microglial superoxide production to damage dopaminergic neurons. Journal of Neuroinflammation, 13, 158.

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Pharmacological Inhibitors for Cell Signaling

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Phorbol 12-myristate 13-acetate (PMA, Sigma P1585) was used in the concentration of 1–10 μM. Twenty millimolar of NEM (Sigma E3876) and 100 μg/ml of cycloheximide was used (Sigma C7698). MG132 is Z-Leu-Leu-Leu-al, used in the concentration of 10 μM (Sigma C2211). Chloroquine diphosphate was used in the concentration of 10 μM (Sigma C6628). Protease inhibitor used was EDTA-free Halt protease inhibitor cocktail (Thermo Scientific 87785). 2-D08, a SUMOylation inhibitor III (Calbiochem 505156, EMD-Millipore) was used at the concentration of 6 μM. Transporter inhibitors in MPP⁺ uptake were included: GBR 12909 dihydrochloride (EMD-Millipore 505732), Mazindol (Sigma M2017), Desipramine hydrochloride (Sigma D3900), and Citalopram (Sigma C7861).

Cartier E., Garcia-Olivares J., Janezic E., Viana J., Moore M., Lin M.L., Caplan J.L., Torres G, & Kim Y.H. (2019). The SUMO-Conjugase Ubc9 Prevents the Degradation of the Dopamine Transporter, Enhancing Its Cell Surface Level and Dopamine Uptake. Frontiers in Cellular Neuroscience, 13, 35.

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Regulation of Dopamine Homeostasis

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Cells were expanded and differentiated for 6 days as described before^{50 (link)}. The effects of NRG1 on extracellular DA levels, as well as on [³H]-Methyl-4-phenylpyridinium (MPP⁺)/[³H]-DA uptake, were measured in absence or presence of the ErbB kinase inhibitor PD158780 (10μM) and the DAT-selective inhibitor GBR12935 (100nM). Extracellular DA content in LUHMES media was determined by HPLC as described above. [³H]-MPP⁺/[³H]-DA uptake assays were performed as described previously^{51 (link)}. Briefly, cells were incubated for 20min with NRG1 and/or PD158780 in DMEM/F12 at 37°C before the addition of 20nM [³H]-DA (30.0Ci/mmol; Perkin Elmer) or [³H]-MPP⁺ (79.8Ci/mmol; Perkin Elmer). After 10min, cells were washed with ice-cold PBS and lysed with 1% SDS. Non-specific uptake was determined with 0.1mM mazindol (Sigma-Aldrich). The accumulated labeled substrate was measured with a LSC6000 counter (Beckman Coulter, Brea, CA).

Skirzewski M., Karavanova I., Shamir A., Erben L., Garcia-Olivares J., Shin J.H., Vullhorst D., Alvarez V.A., Amara S.G, & Buonanno A. (2017). ErbB4 signaling in dopaminergic axonal projections increases extracellular dopamine levels and regulates spatial/working memory behaviors. Molecular Psychiatry, 23(11), 2227-2237.

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Quantifying Dopamine Transporter Activity

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Functional dopamine active transporter (DAT) activity was quantified by incubating differentiated cultures in the presence of ³H-DA (10 nM, GE Healthcare). Briefly, neurons were washed once with PBS prior to addition of ³H-DA either in the presence or absence of mazindol (10 µM, Sigma). For each experimental well, 1 ml of 3H-DA mixture was added for the required time before removal. Uptake was stopped by the addition of ice-cold PBS followed by lysis in sodium hydroxide (1 M). A small sample was retained for protein analysis whilst the remainder was diluted with 5 ml scintillation fluid (OptiPhase SuperMix, Perkin-Elmer) and quantified using a scintillation counter (Beckman Coulter).

Hartfield E.M., Yamasaki-Mann M., Ribeiro Fernandes H.J., Vowles J., James W.S., Cowley S.A, & Wade-Martins R. (2014). Physiological Characterisation of Human iPS-Derived Dopaminergic Neurons. PLoS ONE, 9(2), e87388.

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Lipid-based Nanoparticle Synthesis and Characterization

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1,2-dioleoylsn-glycero-3-phosphoethanolamine-n-[poly(ethylene glycol)]2000 (DSPE-PEG2000) ,1,2-dioleylsn-glycero-3-phosphoethanolamine-n-[poly(ethylenenglycol)]2000-maleimide(DSPE-PEG2000-Mal) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-[poly(ethyleneglycol)]-hydroxy succinamide (DSPE-PEG-NHS,PEG Mw = 2,000 Da) were purchased from Shanghai Advance Vehicle Technology Pharmaceutical L.T.D (Shanghai, China). Cell-penetrating peptide B6 was from GL Biochem Ltd. Co (Shanghai, China). (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich. Epigallocatechin-3-gallate and Mazindol were purchased from Sigma. 4'-Hydroxy phenylboronic acid was purchased from Solarbio Science and Technology co. L.T.D (Beijing, China). Cell culture products were purchased from Gibco-BRL-Life Technologies (UK). Western Blot products were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Li Y., Chen Z., Lu Z., Yang Q., Liu L., Jiang Z., Zhang L., Zhang X, & Qing H. (2018). “Cell-addictive” dual-target traceable nanodrug for Parkinson's disease treatment via flotillins pathway. Theranostics, 8(19), 5469-5481.

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Dopamine Uptake Assay Protocol

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Uptake assays were performed by incubating the cultures with 1 μM [³H]-DA (PerkinElmer Life Sciences, Santa Clara, CA, USA) for 20 min at 37°C, as previously described (Gao et al. 2002 (link)). Nonspecific uptake was determined in the presence of 10 μM mazindol (Sigma-Aldrich, St. Louis, MO, USA).

Wang Q., Chu C.H., Oyarzabal E., Jiang L., Chen S.H., Wilson B., Qian L, & Hong J.S. (2014). Subpicomolar diphenyleneiodonium inhibits microglial NADPH oxidase with high specificity and shows great potential as a therapeutic agent for neurodegenerative diseases. Glia, 62(12), 2034-2043.

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Immunohistochemical Analysis of Neuroinflammation

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Poly-d-lysine, cytosine β-d-arabinofuranoside (Ara-c), l-leucine methyl ester (LME) and 3,3′-diaminobenzidine, mazindol, and urea-hydrogen peroxide tablets were purchased from Sigma-Aldrich (St. Louis, MO, USA). LPS (E. coli strain O111:B4) and NADPH oxidase inhibitor DPI were purchased from Sigma (San Diego, CA, USA). Cell culture ingredients were obtained from Life Technologies (Grand Island, NY, USA). Antibody diluents were purchased from DAKO (Carpinteria, CA, USA). Anti-complement C3 antibody, Complement C3, and iC3b protein (endotoxin-free) were purchased from Millipore Sigma, and the anti-Iba1 antibody was purchased from Wako Pure Chemicals (Richmond, VA, USA). Anti-3-nitrotyrosine (3-NT) and anti-tyrosine hydroxylase (TH) antibodies were purchased from Abcam (Cambridge, MA, USA). TNF-α ELISA kit and isotype control antibodies were purchased from R&D Systems (Minneapolis, MN, USA). Alexa Fluor 594 donkey anti-rabbit IgG and Alexa Fluor 488 donkey anti-goat IgG were purchased from cell signaling (Burlingame, CA, USA).

Zhou R., Chen S.H., Zhao Z., Tu D., Song S., Wang Y., Wang Q., Feng J, & Hong J.S. (2023). Complement C3 Enhances LPS-Elicited Neuroinflammation and Neurodegeneration Via the Mac1/NOX2 Pathway. Molecular Neurobiology, 60(9), 5167-5183.

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Measurement of Dopamine Uptake and Release

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Cells were expanded and differentiated for 6 days as described before.^{50 (link)} The effects of NRG1 on extracellular DA levels, as well as on [³H]-Methyl-4-phenylpyridinium (MPP⁺)/[³H]-DA uptake, were measured in absence or presence of the ErbB kinase inhibitor PD158780 (10 μm) and the DAT-selective inhibitor GBR12935 (100 nm). Extracellular DA content in Lund Human Mesencephalic (LUHMES) media was determined by HPLC as described above. [³H]-MPP⁺/[³H]-DA uptake assays were performed as described previously.^{51 (link)} In brief, cells were incubated for 20 min with NRG1 and/or PD158780 in DMEM/F12 at 37 °C before the addition of 20 nm [³H]-DA (30.0 Ci mmol⁻¹; Perkin Elmer) or [³H]-MPP⁺ (79.8 Ci mmol⁻¹; Perkin Elmer). After 10 min, cells were washed with ice-cold phosphate-buffered saline and lysed with 1% sodium dodecyl sulfate. Non-specific uptake was determined with 0.1 mm mazindol (Sigma-Aldrich, St. Louis, MO, USA). The accumulated labeled substrate was measured with a LSC6000 counter (Beckman Coulter, Brea, CA, USA).

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Neuroinflammation Modulation Assay Protocol

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Xanthine, Xanthine oxidase, SQ 22,536, L798106, poly-D-lysine, superoxide dismutase (SOD), mazindol, 3,3′-diaminobenzidine and urea-hydrogen peroxide tablets were purchased from Sigma-Aldrich (St. Louis, MO, USA). Water-soluble tetrazolium salt-1 (WST-1) was purchased from Dojindo (Rockville, MD, USA). Lipopolysaccharide (LPS; E. coli strain O111: B4) was purchased from Calbiochem (San Diego, CA, USA). Cell culture ingredients were obtained from Life Technologies (Grand Island, NY, USA). Prostaglandin E₂ (PGE₂), 17-phenyl trinor prostaglandin E₂, butaprost, CAY-10598, SC-19220, AH-6809 and AH-23848 were purchased from Cayman Chemical (Ann Arbor, MI, USA). Anti-tyrosine hydroxylase (TH) was purchased from Chemicon (Billerica, MA, USA) and antibody diluent was purchased from DAKO (Carpinteria, CA, USA). TNF-α ELISA kit was purchased from R&D Systems (Minneapolis, MN, USA). Direct cAMP ELISA kit was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Anti-p47^phox antibody was purchased from Millipore (Temecula, CA, USA). Anti-gp91^phox antibody was purchased from BD Biosciences (San Jose, CA, USA). Anti-GAPDH antibody was purchased from Abcam (Cambridge, MA, USA). Alexa Fluor 488 goat anti-rabbit IgG was purchased from Invitrogen (Carlsbad, CA, USA). Goat anti-rabbit biotinylated secondary antibody was purchased from Vector Laboratory (Burlingame, CA, USA).

Chen S.H., Sung Y.F., Oyarzabal E.A., Tan Y.M., Leonard J., Guo M., Li S., Wang Q., Chu C.H., Chen S.L., Lu R.B, & Hong J.S. (2018). Physiological concentration of prostaglandin E2 exerts anti-inflammatory effects by inhibiting microglial production of superoxide through a novel pathway. Molecular neurobiology, 55(10), 8001-8013.

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