3h mpp

Manufactured by PerkinElmer

Sourced in United States

[3H]-MPP+ is a radioactively labeled compound commonly used in research applications. It is a derivative of the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), with the addition of a tritium (3H) label. This product is intended for use in scientific research, where it can serve as a tool for investigating various biological processes and pathways.

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Lab products found in correlation

Mazindol, by Merck Group (2 mentions) Lsc6000 counter, by Beckman Coulter (2 mentions) Analytical grade reagents, by Merck Group (1 mentions) 14c sucrose, by PerkinElmer (1 mentions) 3h l phenylalanine, by PerkinElmer (1 mentions) Complete mini cocktail, by Roche (1 mentions) Mass spectrometry grade rlys c, by Promega (1 mentions) Tetraethyl ammonium tea, by Merck Group (1 mentions) Sodium dodecyl sulfate (sds), by Merck Group (1 mentions) Sequencing grade modified trypsin, by Promega (1 mentions) Proteasemax surfactant, by Promega (1 mentions) Liquid scintillation counter, by Beckman Coulter (1 mentions) 4 phenylbutyric acid, by Merck Group (1 mentions) Ultima gold scintillation mixture, by PerkinElmer (1 mentions) Cell culture media, by Merck Group (1 mentions)

6 protocols using 3h mpp

Radiotracer Quantification Protocol

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[¹⁴C]-Sucrose (580–600 mCi/mmol), [³H]-L-phenylalanine (128 Ci/mmol) and [³H]-MPP⁺ (70–84 Ci/mmol) were purchased from Perkin Elmer (Paris, France). Protease Inhibitor Cocktail Complete Mini^® was purchased from Roche (Basel, Switzerland). Protease MAX surfactant, mass spectrometry grade rLys-C and sequencing grade modified trypsin were acquired from Promega (Charbonnières-les-Bains, France). RIPA buffer was prepared employing analytical grade reagents from Sigma Aldrich (Saint Quentin Fallavier, France): 50 mmol L⁻¹ Tris (pH 8.0), 150 mmol L⁻¹ NaCl, 1% (V/V) Triton X-100, 0.1% (V/V) sodium dodecyl sulfate (SDS) and 0.5% (W/V) sodium deoxycholate in high purity water. Standard peptides for protein quantification were provided by Bertin Pharma (Orleans, France). Tetraethylammonium (TEA) was purchased from Sigma Aldrich. All other chemicals were of analytical grade.

Chaves C., Campanelli F., Chapy H., Gomez-Zepeda D., Glacial F., Smirnova M., Taghi M., Pallud J., Perrière N., Declèves X., Menet M.C, & Cisternino S. (2020). An Interspecies Molecular and Functional Study of Organic Cation Transporters at the Blood-Brain Barrier: From Rodents to Humans. Pharmaceutics, 12(4), 308.

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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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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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Monoamine Transporter Inhibition Assay

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N27 cells were plated in a 24-well plate pre-coated with poly-D-Lysine. Cells were incubated with [³H]-methyl-4-phenylpyridinium (MPP⁺) at 37°C for 15 min. For uptake experiments, cells were incubated with specific DAT inhibitors: GBR12935 (10 μM) and mazindol (100 μM), NET inhibitor: desipramine (10 μM), and SERT inhibitor: citalopram (10 μM). All monoamine inhibitors were preincubated for 10 min before the addition of 50 nM of [³H]-MPP⁺ (79.8 Ci/mmol, Perkin Elmer) in RPMI cell culture media, and were maintained during the uptake. The uptake of [³H]-MPP⁺ was blocked after 15 min in ice-cold NaCl-free uptake buffer. Cells were gently washed twice and lysed in 0.4 ml of 1% SDS. The accumulated [³H]-MPP⁺ was measured by a liquid scintillation counter (Beckman).

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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Radiolabeled MPP+ Uptake Assay

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[³H]MPP⁺ (N-methyl-4-phenylpyridinium, 82.9 Ci/mmol) and ULTIMA Gold scintillation mixture were purchased from PerkinElmer Life Science (Waltham, MA, USA). Decynium-22 was ordered from Synthon Chemicals (Bitterfeld, Wolfen, Germany). All other chemicals, including 4-phenyl butyric acid, and cell culture media were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Sarstedt (Nuembrecht, Germany).

Angenoorth T.J., Maier J., Stankovic S., Bhat S., Sucic S., Freissmuth M., Sitte H.H, & Yang J.W. (2022). Rescue of Misfolded Organic Cation Transporter 3 Variants. Cells, 12(1), 39.

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Trospium Transport Kinetics Assessment

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All chemicals, unless otherwise stated, were obtained from Sigma-Aldrich (Taufkirchen, Germany). [³H]MPP⁺ (80 Ci/mmol) was obtained from PerkinElmer (Waltham, MA, USA) and [³H]trospium trifluoroacetate (24.6 Ci/mmol) was obtained from RC Tritec AG (Teufen, Switzerland). Unlabeled trospium chloride was kindly provided by Dr. Pfleger Arzneimittel GmbH (Bamberg, Germany). For the transport experiments, [³H]trospium trifluoroacetate was mixed with an excess of unlabeled trospium chloride. Due to the high excess of chloride in relation to trifluoroacetate in this preparation [³H]trospium chloride is regarded as the active compound. The mixture of [³H]trospium and unlabeled trospium chloride used for all transport measurements is referred to as trospium in the manuscript.

Gorecki M., Müller S.F., Leidolf R, & Geyer J. (2020). Trospium Chloride Transport by Mouse Drug Carriers of the Slc22 and Slc47 Families. International Journal of Molecular Sciences, 22(1), 22.

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