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3h raclopride

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[3H]-raclopride is a radioligand used for the in vitro and in vivo quantification of dopamine D2 receptors. It has a high affinity and selectivity for the dopamine D2 receptor.

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Lab products found in correlation

Butaclamol, by Merck Group (5 mentions) 3h sch23390, by PerkinElmer (4 mentions) Multiscreen vacuum manifold 96 well, by Merck Group (3 mentions) Durapore membrane, by Merck Group (2 mentions) 3h mazindol, by PerkinElmer (2 mentions) Sch23390, by Merck Group (1 mentions) Wallac 1450 microbeta trilux, by PerkinElmer (1 mentions) Dc protein assay, by Bio-Rad (1 mentions) Butaclamol hydrochloride, by Merck Group (1 mentions) Sulpiride, by Bio-Techne (1 mentions) Desipramine hydrochloride, by Bio-Techne (1 mentions) Bacitracin, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Liquid nicotine, by Merck Group (1 mentions) 125i epibatidine, by PerkinElmer (1 mentions) 3h spiperone, by PerkinElmer (1 mentions) Ultima gold scintillation cocktail, by PerkinElmer (1 mentions) Pcdna3l his camyel, by American Type Culture Collection (1 mentions) Alphascreen reagents, by PerkinElmer (1 mentions) 384 well proxiplate, by PerkinElmer (1 mentions)

12 protocols using 3h raclopride

1

Saturation Binding of [3H]-Raclopride in HEK Cells

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Saturation binding experiments with the D2-likeR antagonist [3H]-raclopride (specific activity 82.8 Ci/mmol, PerkinElmer Life Sciences, Sweden) were performed in membrane preparations from single and cotransfected HEK cells (100 μg protein/ml) incubated with increasing concentrations of [3H]-raclopride (ranging from 0.1 nM to 12 nM) in 250 μl of incubation buffer (50 mM Tris–HCl, 100 mM NaCl, 7 mM MgCl2, 1 mM EDTA, 0.05% BSA and 1 mM dithiothreitol) for 60 min at 30 °C in the presence or absence of cocaine (1 nM, 10 nM, and 100 nM) and the high affinity sigma 1 receptor antagonist PD144418 [26 (link)]. Non-specific binding was defined by radioligand binding in the presence of 10 μM (+) butaclamol (Sigma Aldrich, Sweden). The incubation was terminated by rapid filtration Whatman GF/B filters (Millipore Corp, Sweden) using a MultiScreen™ Vacuum Manifold 96-well followed by three washes (~ 250 μl per wash) with ice-cold washing buffer (50 mM Tris–HCl pH 7.4). The filters were dried, 5 ml of scintillation cocktail was added, and the bound ligand was determined after 12 h by liquid scintillation spectrometry.
Borroto-Escuela D.O., Narváez M., Romero-Fernández W., Pinton L., Wydra K., Filip M., Beggiato S., Tanganelli S., Ferraro L, & Fuxe K. (2019). Acute Cocaine Enhances Dopamine D2R Recognition and Signaling and Counteracts D2R Internalization in Sigma1R-D2R Heteroreceptor Complexes. Molecular Neurobiology, 56(10), 7045-7055.
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2

Receptor Binding Density Quantification

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The receptor binding density for D1 and D2 receptors was adapted from the protocols described in Sommer et al. (31 (link)) and Behuet et al.’s study (32 (link)). The tritiated ligands [3H]-SCH23390 and [3H]-Raclopride were purchased from PerkinElmer (Germany). SCH23390 is a potent D1 and D5 receptor antagonist. Raclopride is a D2 and D3 selective receptor antagonist. Both ligands bind to the receptors localized on the cell surface, without penetrating inside the cell membrane. In the first step, the pre-incubation, endogenous ligands were washed off. In the following main incubation, the tritiated ligands were incubated both in the presence of a competitor, in order to determine the unspecific binding, and without it, in order to assess the total binding. Finally, the slices were rinsed. The slices incubated with [3H]-Raclopride were additionally dried with a cold air stream. A detailed description of the protocols used is reported in Table 1.
Chhabra S., Nardi L., Leukel P., Sommer C.J, & Schmeisser M.J. (2023). Striatal increase of dopamine receptor 2 density in idiopathic and syndromic mouse models of autism spectrum disorder. Frontiers in Psychiatry, 14, 1110525.
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3

Quinpirole-Raclopride Binding Assay

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Competition experiments of quinpirole (0.3 nM-3 mM) versus the D2-likeR antagonist [3H]-raclopride (2 nM; specific activity 78.1 Ci/mmol, PerkinElmer Life Sciences, Stockholm, Sweden) were carried out by membrane (20 μg per well) incubation at 30 °C for 90 min. Non-specific binding was defined by radioligand binding in the presence of 10 μM (+)-butaclamol (Sigma-Aldrich, Stockholm, Sweden). The incubation was terminated by rapid filtration through hydrophilic (LPB) Durapore ®Membrane (Millipore, Stockholm, Sweden) using a MultiScreen™ Vacuum Manifold 96-well (Millipore Corp, Bedford, MA), followed by five washes (200 μl per wash) with ice-cold washing buffer (50 mM Tris–HCl pH 7.4). The filters were dried, 4 ml of scintillation cocktail was added, and the bound ligand was determined after 12 h by liquid scintillation spectrometry.
Borroto-Escuela D.O., Narváez M., Romero-Fernández W., Pinton L., Wydra K., Filip M., Beggiato S., Tanganelli S., Ferraro L, & Fuxe K. (2019). Acute Cocaine Enhances Dopamine D2R Recognition and Signaling and Counteracts D2R Internalization in Sigma1R-D2R Heteroreceptor Complexes. Molecular Neurobiology, 56(10), 7045-7055.
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4

Competitive Binding Assay for D2-like Receptors

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Competition experiments of quinpirole (0.3 nM–3 mM) versus the D2-likeR antagonist [3H]-raclopride (2 nM; specific activity 78.1 Ci/mmol, PerkinElmer Life Sciences, Stockholm, Sweden) were carried out by membrane (20 μg per well) incubation at 30 °C for 90 min, in the presence or absence of 100 nM of the A2AR agonist CGS 21680. Nonspecific binding was defined by radioligand binding in the presence of 10 μM (+)-butaclamol (Sigma-Aldrich, Stockholm, Sweden). The incubation was terminated by rapid filtration through Hydrophilic (LPB) Durapore ®Membrane (Millipore, Stockholm, Sweden) using a MultiScreen™ Vacuum Manifold 96-well (Millipore Corp, Bedford, MA), followed by five washes (200 μl per wash) with ice-cold washing buffer (50 mM Tris-HCl pH 7.4). The filters were dried, 4 ml of scintillation cocktail was added, and the bound ligand was determined after 12 h by liquid scintillation spectrometry.
Borroto-Escuela D.O., Wydra K., Li X., Rodriguez D., Carlsson J., Jastrzębska J., Filip M, & Fuxe K. (2018). Disruption of A2AR-D2R Heteroreceptor Complexes After A2AR Transmembrane 5 Peptide Administration Enhances Cocaine Self-Administration in Rats. Molecular Neurobiology, 55(8), 7038-7048.
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5

Radiolabeling D1 and D2 Receptors

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D1 and D2 receptors were radiolabeled as previously described (Hersi et al., 1995 (link)). Briefly, slides were incubated with ligands diluted in Tris buffer at room temperature for 60 minutes. For D1 receptor, total radioligand binding was determined using [3H]-SCH 23390 (1 nM; Perkin Elmer, Waltham, MA, USA). To measure the degree of non-specific binding, the contiguous sections were incubated in the same buffer, but supplemented with 1 µM unlabeled SCH23390 (Sigma-Aldrich, St. Louis, MO, USA). D2 receptor binding was determined using [3H]-raclopride (2.85 nM; Perkin Elmer). Non-specific binding of the D2 radioligand was assessed in adjacent sections that were incubated in the same buffer supplemented with 1 µM (+)-butaclamol (Sigma-Aldrich). Test cassettes containing slices from each age and genotype, each with its own [3H] standard, were used to ensure proper exposure time and signal strength. Representative autoradiograms are shown in Fig. 1.
Pokinko M., Grant A., Shahabi F., Dumont Y., Manitt C, & Flores C. (2017). Dcc Haploinsufficiency Regulates Dopamine Receptor Expression Across Postnatal Lifespan. Neuroscience, 346, 182-189.
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6

Dopamine Receptor Binding Assay

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Cells were seeded in poly-D-lysine treated 24-well plates and grown until 50–80% confluent. Cells were then incubated for 30 minutes at 37 °C with assay buffer (DMEM + 5 mg/ml BSA), followed by 30 minutes at 37 °C with [3H]-raclopride (Perkin Elmer, MA, USA) at 1.3 nM, with or without 10 μM unlabelled raclopride as a displacer. Assay buffer was removed and cells were washed twice in ice-cold phosphate buffered saline, then lysed in 0.1 M NaOH for 10–20 minutes. Lysate samples were then mixed with scintillation fluid and scintillation events were measured for 3 minutes per sample in a Wallac 1450 MicroBeta TriLux (Perkin Elmer, MA, USA). Lysate samples were also assayed in DC protein assay (Biorad, CA, USA) to enable normalisation to protein concentration.
Hunter M.R., Grimsey N.L, & Glass M. (2016). Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model. Scientific Reports, 6, 27316.
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7

Detailed D2 Receptor Binding Assay

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D2R binding procedures began with pre-incubation for 30 min at RT in 50 mM TrisHCl buffer, containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2 and 0.001% ascorbic acid (pH 7.4). Incubation for 1 h in 5 nM [3H]Raclopride (specific activity 76 Ci/mmol; PerkinElmer, Waltham, MA, USA) at RT was then used for total D2 receptor binding. Non-specific binding was determined with addition of 10 µM (+)-Butaclamol (Sigma-Aldrich, Castle Hill, NSW, Australia) to subsequent sections. Following incubation, sections were washed twice for 5 min in ice-cold buffer, dipped in ice-cold distilled water and then air dried [40 (link),85 (link),88 (link),89 (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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8

Synthesis and Radiolabeling of Sigma-1 Receptor Ligands

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8-triazaspiro(4.5)decan-4-one,8-(3-(p-fluorobenzoyl)propyl)-1-phenyl-3 hydrochloride (spiperone), 1H-Benzo(6,7)cyclohepta(1,2,3-de)pyrido(2,1-a)isoquinolin-3-ol, 2,3,4,4a,8,9,13b,14-octahydro-3-(1,1-dimethylethyl)-(3S-(3-alpha,4a-alpha,13b-beta)) ((+)-butaclamol) hydrochloride and S-(−)-3-chloro-5-ethyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-6-hydroxy-2-methoxybenzamide hydrochloride (eticlopride), were purchased from Sigma-Aldrich. The synthesis and purification of the SV series of compounds, including 1-((5-methoxy-1H-indol-3-yl)methyl)-4-(4-(methylthio)phenyl)piperidin-4-ol (SV 293), have been previously reported (Vangveravong et al., 2006 (link); Vangveravong et al., 2011). The synthesis and purification of the 4-(dimethylamino)-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)benzamide (WC-10) and N-[(4-[4-[2-(2-fluoroethoxy)phenyl]-1-piperazinyl]butyl]-4-(3-thienyl)benzamide (LS-3-134) have been previously reported (Tu et al., 2011a (link)).
LS-3-134 was radiolabeled with tritium ([3H]LS-3-134) by American Radiolabeled Chemicals, Inc. (St. Louis, MO) to a specific activity of 60 Ci/mmol. [125I]IABN was prepared and purified as previously described (Luedtke et al., 2000 (link)). [3H]raclopride was obtained commercially (NEN-PerkinElmer).
Rangel-Barajas C., Malik M., Taylor M., Neve K.A., Mach R.H, & Luedtke R.R. (2014). Characterization of [3H]LS-3-134, a Novel Arylamide Phenylpiperazine D3 Dopamine Receptor Selective Radioligand. Journal of neurochemistry, 131(4), 418-431.
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9

Radioligand Binding Assay Protocols

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[3H]SCH23390 (specific activity 60 Ci/mmol, KD = 0.7 nM, Bmax = 347 fmol/mg according to Schulz et al. (1985 (link))), [3H]Raclopride (specific activity 80 Ci/mmol, KD = 2.08 nM, Bmax = 20.0 fmol/mg according to Hall et al. (1990 (link))) and [3H]Mazindol (specific activity 17.8 Ci/mmol, KD = 18.2 nM, Bmax = 0.0073 fmol/mg (Javitch et al., 1984 (link))) were from PerkinElmer (Massachusetts, USA), bacitracin, bovine serum albumin, ascorbin acid, nomifensine maleate salt from Sigma-Aldrich (St. Louis, MO, USA), and desipramine hydrochloride, SKF, sulpiride from Tocris Biosciences (Bristol, UK). All other reagents were of analytical grade from regular suppliers.
Sommer W.H., Costa R.M, & Hansson A.C. (2014). Dopamine systems adaptation during acquisition and consolidation of a skill. Frontiers in Integrative Neuroscience, 8, 87.
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10

Radioligand Binding Assay Protocols

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[125I]-Epibatidine (2200 Ci/mmol), [3H]SCH23390 (N-methyl [3H], 84.3 Ci/mmol), [3H]mazindol (4’-[3H], 20.6 Ci/mmol) and [3H]raclopride (methoxy-[3H], 20.6 Ci/mmol) were obtained from Perkin-Elmer NEN, Boston, MA. α-Conotoxin MII (α-CtxMII) and [125I]-α-CtxMII were prepared as described previously (Whiteaker et al. 2000 (link), Cartier et al. 1996 (link)). Unlabeled 5I-epibatidine was a generous gift from Dr. Kenneth Kellar, Georgetown University, Washington, DC. Liquid (−)-nicotine was purchased from Sigma-Aldrich Chemical Company (St. Louis, MO) and was redistilled periodically. All additional chemicals were of reagent grade.
Marks M., Grady S., Salminen O., Paley M., Wageman C., McIntosh J, & Whiteaker P. (2014). α6β2*-subtype nicotinic acetylcholine receptors are more sensitive than α4β2*-subtype receptors to regulation by chronic nicotine administration. Journal of neurochemistry, 130(2), 185-198.
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