3h win35 428

Manufactured by PerkinElmer

Sourced in United States

[3H]WIN35,428 is a radioactive compound that is used as a research tool in scientific studies. It is a radioligand that binds to specific receptors or proteins of interest in biological systems. The core function of [3H]WIN35,428 is to serve as a labeled probe for the detection and quantification of these targets in various experimental settings.

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16 protocols using 3h win35 428

Radioligand Binding Assay for Dopamine Transporter

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Competition binding experiments were carried out incubating 8 nM [³H]-WIN 35,428 (specific activity 84 Ci/mmol; Perkin Elmer, Boston, MA, USA) with CHO membranes transfected with human DAT (Perkin Elmer) or mouse striatal synaptosomes with different concentration of the examined compounds for 120 min at 4°C. Non-specific binding was determined in the presence of 1 µM GBR 12783. At the end of the incubation time, bound and free radioactivity were separated by filtering the assay mixture through Whatman GF/B glass fiber filters in a Brandel cell harvester (Brandel, Unterföhring, Germany). Filter bound radioactivity was counted in a Perkin Elmer 2810TR scintillation counter (Perkin Elmer).

Ossato A., Uccelli L., Bilel S., Canazza I., Di Domenico G., Pasquali M., Pupillo G., De Luca M.A., Boschi A., Vincenzi F., Rimondo C., Beggiato S., Ferraro L., Varani K., Borea P.A., Serpelloni G., De-Giorgio F, & Marti M. (2017). Psychostimulant Effect of the Synthetic Cannabinoid JWH-018 and AKB48: Behavioral, Neurochemical, and Dopamine Transporter Scan Imaging Studies in Mice. Frontiers in Psychiatry, 8, 130.

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Radioligand Binding Assay for DAT

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DAT binding assays were performed using 2 nM [³H]WIN
35,428 (specificity activity, 84.5 Ci mmol^–1; PerkinElmer
Life Science) as previously described by Nencetti et al.^{29 (link)}

Ortore G., Orlandini E., Betti L., Giannaccini G., Mazzoni M.R., Camodeca C, & Nencetti S. (2020). Focus on Human Monoamine Transporter Selectivity. New Human DAT and NET Models, Experimental Validation, and SERT Affinity Exploration. ACS Chemical Neuroscience, 11(20), 3214-3232.

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Quantifying Dopamine Receptor Changes in Amphetamine-Treated Rats

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Animals (N = 12 AMPH pre-treated, N = 12 saline pre-treated) were sedated with isoflurane and euthanized by cervical dislocation and the brains were rapidly removed, snap frozen and stored at -80°C. Frontal brain sections (14 μm) were cut and DAT and DRD1 autoradiography was performed on the CPu and NAcc. [³H]WIN35428 (Perkin-Elmer^®, France; specific radioactivity = 3.034 MBq/nmol; 5 concentrations from 0.55 to 15.0 nM) was used for the DAT binding experiments according to protocols described before by Hebert [24 (link)]. Non-specific binding was determined by incubation of adjacent brain slices in the presence of 10 μM nomifensine. For the DRD1 binding experiments, [³H]SCH-23,390 (Perkin-Elmer^®, France; specific radioactivity = 3.119 MBq/nmol; 5 concentrations from 0.10 to 8.1 nM) was used and performed according to the Savasta protocol [25 (link)]. Non-specific binding was determined by incubation of adjacent brain slices in the same conditions and in the presence of 10 μM SKF38393. Brain sections were exposed to tritium-sensitive phosphor imaging plates (Perkin-Elmer^®) before acquisition of images (Cyclone^®, Perkin-Elmer^®). Specific binding was calculated as the difference between total and non-specific binding and K_d and B_max values were derived from raw data using nonlinear fitting procedures (Prism^®).

Schrantee A., Tremoleda J.L., Wylezinska-Arridge M., Bouet V., Hesseling P., Meerhoff G.F., de Bruin K.M., Koeleman J., Freret T., Boulouard M., Desfosses E., Galineau L., Gozzi A., Dauphin F., Gsell W., Booij J., Lucassen P.J, & Reneman L. (2017). Repeated dexamphetamine treatment alters the dopaminergic system and increases the phMRI response to methylphenidate. PLoS ONE, 12(2), e0172776.

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Quantifying Dopamine Transporter Binding

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Brain sections for DAT binding were pre-incubated in 50 mM Tris-HCl buffer containing 120 mM NaCl and 0.1% bovine serum albumin (pH 7.4) for 20 min at 4 °C. Sections were then incubated for 2 h in 15 nM [³H]WIN35428 (specific activity, 85.9 Ci/mmol; PerkinElmer, Waltham, MA, USA). Non-specific binding was determined by the addition of 10 µM GBR12909 (Sigma-Aldrich, Castle Hill, NSW, Australia) to subsequent sections. Sections were then washed twice for 1 min in ice-cold buffer, dipped in ice-cold distilled water and then air dried [40 (link),85 (link),88 (link)].

De Santis M., Lian J., Huang X.F, & Deng C. (2016). Early Antipsychotic Treatment in Juvenile Rats Elicits Long-Term Alterations to the Dopamine Neurotransmitter System. International Journal of Molecular Sciences, 17(11), 1944.

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Radioligand Binding Assay for Dopamine Transporter

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Frozen striatum membranes^{33 (link)} dissected from male Sprague-Dawley rat brains (supplied on ice by Bioreclamation, Hicksville, NY) were homogenized in 20 volumes (w/v) of ice cold modified sucrose phosphate buffer (0.32 M sucrose, 7.74 mM Na₂HPO₄, and 2.26 mM NaH₂PO₄, pH adjusted to 7.4) using a Brinkman Polytron (Setting 6 for 20 s) and centrifuged at 48,400 × g for 10 min at 4°C. The resulting pellet was resuspended in buffer, recentrifuged, and suspended in ice cold buffer again to a concentration of 20 mg/mL, original wet weight (OWW). Experiments were conducted in 96-well polypropylene plates containing 50 μL of various concentrations of the inhibitor, diluted using 30% DMSO vehicle, 300 μL of sucrose phosphate buffer, 50 μL of [³H]-WIN35,428 (final concentration 1.5 nM; PerkinElmer Life Sciences, Waltham, MA), and 100 μL of tissue (2.0 mg/well OWW). All compound dilutions were tested in triplicate and the competition reactions started with the addition of tissue, and the plates were incubated for 120 min at 0–4°C. Nonspecific binding was determined using 10 μM indatraline.

Giancola J.B., Bonifazi A., Cao J., Ku T., Haraczy A.J., Lam J., Rais R., Coggiano M.A., Tanda G, & Newman A.H. (2020). Structure-Activity Relationships for a Series of (Bis(4-fluorophenyl)methyl)sulfinylethyl-aminopiperidines and -piperidine amines at the Dopamine Transporter: Bioisosteric Replacement of the Piperazine Improves Metabolic Stability. European journal of medicinal chemistry, 208, 112674.

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Radioligand Binding Assay Protocol

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Albiflorin, obtained from Beijing Wonner Biotech Co., Ltd. (China), has a molecular formula of C₂₃H₂₈O₁₁ and a molecular weight of 480.46. [³H]-citalopram, [³H]-WIN35,428, [³H]-nisoxetine, [³H]5-HT, [³H]-NE and [³H]-DA were purchased from PerkinElmer Life Sciences (NEN, Boston, MA). Scintillation cocktail (Ultima Gold, catalogue number 6013329) was purchased from PerkinElmer Life and Analytical Sciences. Dulbecco’s modified Eagle’s medium and foetal bovine serum were purchased from Invitrogen Inc. (Grand Island, NY) and HyClone Corp. (South Logan, UT), respectively. Desipramine (catalogue number D-3900), fluoxetine (catalogue number F-132), paroxetine (catalogue number P-1372) and nomifensine (catalogue number M-2017) were purchased from Sigma-Aldrich (St. Louis, MO). All other radioligands were purchased from PerkinElmer Life and Analytical Sciences.

Jin Z.L., Gao N., Xu W., Xu P., Li S., Zheng Y.Y, & Xue M. (2016). Receptor and transporter binding and activity profiles of albiflorin extracted from Radix paeoniae Alba. Scientific Reports, 6, 33793.

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Putamen DAT Binding Assay

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DAT binding assays were done as described previously (Mash et al., 2002 (link)). Briefly, ventromedial putamen (200 mg) was dissected from cryopreserved brain specimens. Tissues were homogenized in 10 mM Na-Phosphate/0.32 M sucrose buffer with Brinkman Polytron at setting 2.5 for 15 s and centrifuged at 32,000× g for 15 min. The membrane pellet was washed once and re-suspended in phosphate-sucrose buffer at a dilution of 1:20 (w/v). 0.5 nM [³H]WIN35,428 (0.5 nM, 84Ci/mmol, PerkinElmer) and incubated with 5 mg/well membranes in the presence of increasing concentrations of unlabeled [³H]WIN35,428 (0.1 nM–10 μM) for 2 h at 4°C. The reaction was terminated by rapid filtration through 934AH filters and measured by liquid scintillation counting.

Wu C., Garamszegi S.P., Xie X, & Mash D.C. (2017). Altered Dopamine Synaptic Markers in Postmortem Brain of Obese Subjects. Frontiers in Human Neuroscience, 11, 386.

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Synthesis and Characterization of N-Ethyl Cathinones

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N-Ethyl cathinones were synthesized in a racemic form as hydrochloride
salts as described in Section 3.3. Solutions for injection were prepared
daily in an isotonic saline solution (0.9% NaCl, pH 7.4). Cocaine
and methamphetamine hydrochloride were generously provided by the
Spanish National Institute of Toxicology and Dr. Riera laboratory
from Parc Científic de Barcelona, respectively. α-PVP·HCl
was synthesized as described in ref (6 (link)). Cell culture media [Dulbecco’s modified
Eagle’s medium (DMEM) high-glucose] was purchased from Sigma-Aldrich.
[³H]1-Methyl-4-phenylpyridinium ([³H]MPP⁺) was supplied by American Radiolabeled Chemicals (St. Louis,
USA). [³H]5-HT, [³H]imipramine, and [³H]WIN35,428 were purchased from PerkinElmer Inc. (Boston, MA, USA).
The NGF was supplied by Upstate Biotechnology (Lake Placid, NY). The
CCK-8 cell counting kit was purchased from Vazyme Biotech Co., Ltd.
All other reagents were of analytical grade and purchased from several
commercial sources.

Nadal-Gratacós N., Ríos-Rodríguez E., Pubill D., Batllori X., Camarasa J., Escubedo E., Berzosa X, & López-Arnau R. (2023). Structure–Activity Relationship of N-Ethyl-Hexedrone Analogues: Role of the α-Carbon Side-Chain Length in the Mechanism of Action, Cytotoxicity, and Behavioral Effects in Mice. ACS Chemical Neuroscience, 14(4), 787-799.

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Dopamine Transporter Binding Assay

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Striata were dissected from male
Sprague–Dawley rat brains (supplied on ice from Bioreclamation
(Hicksville, NY), prepared by homogenizing tissues in 20 volumes (w/v)
of ice cold modified sucrose phosphate buffer (0.32 M sucrose, 7.74
mM Na₂HPO₄, 2.26 mM NaH₂PO₄, pH adjusted to 7.4) using a Brinkman Polytron (setting 6 for 20
s), and centrifuged at 30000g for 10 min at 4 °C.
The resulting pellet was resuspended in buffer, recentrifuged, and
suspended in buffer again to a concentration of 10 mg/mL, original
wet weight (OWW). Experiments were conducted in assay tubes containing
0.5 mL of sucrose phosphate buffer, 0.5 nM [³H]WIN 35,428
(K_d = 5.53, specific activity 84 Ci/mmol;
Perkin-Elmer Life Sciences, Waltham, MA), 1.0 mg of tissue OWW, and
various concentrations of inhibitor. The reaction was started with
the addition of tissue, and the tubes were incubated for 120 min on
ice. Nonspecific binding was determined using 100 μM cocaine
hydrochloride.

Okunola-Bakare O.M., Cao J., Kopajtic T., Katz J.L., Loland C.J., Shi L, & Newman A.H. (2014). Elucidation of Structural Elements for Selectivity across Monoamine Transporters: Novel 2-[(Diphenylmethyl)sulfinyl]acetamide (Modafinil) Analogues. Journal of Medicinal Chemistry, 57(3), 1000-1013.

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Frozen Striatal Membrane Binding Assay

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Frozen striatal membranes, dissected from male Sprague−Dawley rat brains (supplied on ice by Bioreclamation, Hicksville, NY), were homogenized in 20 volumes (w/v) of ice cold modified sucrose phosphate buffer (0.32 M sucrose, 7.74 mM Na₂HPO₄, and 2.26 mM NaH₂PO₄, pH adjusted to 7.4) using a Brinkman Polytron (Setting 6 for 20 s) and centrifuged at 48,400 × g for 10 min at 4 °C. The resulting pellet was resuspended in buffer, recentrifuged, and suspended in ice cold buffer again to a concentration of 20 mg/mL, original wet weight (OWW). Experiments were conducted in 96-well polypropylene plates containing 50 μL of various concentrations of the inhibitor, diluted using 30% DMSO vehicle, 300 μL of sucrose phosphate buffer, 50 μL of [³H]WIN 35,428 (final concentration 1.5 nM; PerkinElmer Life Sciences, Waltham, MA), and 100 μL of tissue (2.0 mg/well OWW). All compound dilutions were tested in triplicate and the competition reactions started with the addition of tissue, and the plates were incubated for 120 min at 0–4 °C. Nonspecific binding was determined using 10 μM indatraline.

Lee K.H., Fant A.D., Guo J., Guan A., Jung J., Kudaibergenova M., Miranda W.E., Ku T., Cao J., Wacker S., Duff H.J., Newman A.H., Noskov S.Y, & Shi L. (2021). Toward Reducing hERG Affinities for DAT Inhibitors with a Combined Machine Learning and Molecular Modelling Approach. Journal of chemical information and modeling, 61(9), 4266-4279.

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