Microscint 0

Manufactured by PerkinElmer

MicroScint 0 is a liquid scintillation cocktail designed for counting radioactive samples. It provides efficient light output and long-term stability for counting of samples in microplates, vials, and other small-volume containers.

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Lab products found in correlation

Topcount, by PerkinElmer (6 mentions) White 96 well plate, by PerkinElmer (2 mentions) Methyl 3h thymidine, by PerkinElmer (2 mentions) Gf b unifilter plates, by Hewlett-Packard (2 mentions) Topcounter, by PerkinElmer (1 mentions) Prizm 6, by GraphPad (1 mentions) Cell harvester, by PerkinElmer (1 mentions) Pirenzepine, by GraphPad (1 mentions) 3h pirenzepine, by PerkinElmer (1 mentions) Gf c filter plates, by PerkinElmer (1 mentions) 3h pirenzepine, by GraphPad (1 mentions) Prism 5, by GraphPad (1 mentions) Topcount, by Hewlett-Packard (1 mentions) Unifilter 96 gf c, by PerkinElmer (1 mentions) Topcount nxt microplate scintillation counter, by PerkinElmer (1 mentions)

Spelling variants of this product

Microscint-0 scintillation cocktail (3 citations) MicroScint-0 cocktail (2 citations) MicroScint-0 scintillant cocktail (2 citations)

10 protocols using microscint 0

NMDA Receptor Binding Assay with Linalool and Linalyl Acetate

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Affinity for native NMDA receptors was determined using 2 nM [³H]-CGP 39653 (Sills et al., 1991 (link)) with some modification. On the day of the assay, frozen membranes were quickly thawed and homogenized in 30 volumes of ice-cold Tris-HCl buffer, pH 7.4 (50 mM containing 2.5 mM CaCl₂), and centrifuged (48,000 × g for 10 min). This step was repeated three times. The final pellet was re-suspended in ice-cold buffer, corresponding to approximately 0.4–0.5 mg protein/ml. Binding was carried out in aliquots consisting of 25 μL [³H]-CGP 39653, 25 μL test solution, and 200 μL membrane suspension and incubated at 0° for 60 min. Non-specific binding was determined using 1 mM (S)-Glu. Binding was terminated by filtration through Whatman GF/C filters using a 96-well Packard Filter-Mate Cell Harvester and filters were washed with 3 × 250 μL of ice-cold buffer. After drying, 30 μL Microscint 0 (Perkin-Elmer) per well was added and the filter was counted on a Topcounter (Perkin-Elmer). Linalool and linalyl acetate were also tested in this assay.

López V., Nielsen B., Solas M., Ramírez M.J, & Jäger A.K. (2017). Exploring Pharmacological Mechanisms of Lavender (Lavandula angustifolia) Essential Oil on Central Nervous System Targets. Frontiers in Pharmacology, 8, 280.

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Characterization of Gα-Coupled GPCR Activation

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Membranes were prepared from Sf9 cells expressing Gα_sβ₁γ₂ and β₂AR. Membranes (~15 μg) were pretreated with GDP (final assay concentration of 1 μM) in a GTPγS assay buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl₂, and 1 mM ascorbic acid) and different concentrations of agonist (Epi, c-Epi, NorEpi, c-NorEpi, Iso or c-Iso) for 10 min at room temperature before adding [³⁵S]GTPγS (for a final concentration of 0.1 nM). The assay was incubated at 30 ^oC for 30 min before stopping by rapid filtration through GF/B Unifilter plates (Whatman) and washing with ice-cold assay buffer. Filter plates were dried before adding Microscint 0™ and counting bound [³⁵S]GTPγS (Perkin Elmer) using a TopCount™ (Perkin Elmer). Data were analyzed using Prizm 6.0 (GraphPad, LLC, CA). Figures show the combined results from three separate experiments performed in duplicate.

Xu X., Shonberg J., Kaindl J., Clark M.J., Stößel A., Maul L., Mayer D., Hübner H., Hirata K., Venkatakrishnan A.J., Dror R.O., Kobilka B.K., Sunahara R.K., Liu X, & Gmeiner P. (2023). Constrained catecholamines gain β2AR selectivity through allosteric effects on pocket dynamics. Nature Communications, 14, 2138.

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Radioligand Binding Assay for Beta-Adrenergic Receptors

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In a polypropylene 96 well plate, binding assay buffer or propranolol (50 μM final for nonspecific binding), [³H] dihydroalprenolol (2 nM final), and membranes (5 μg per well) were plated in order and incubated for 2 hours at room temperature to reach equilibrium. GF/C filterplates were prepared with 0.3% polyethylenimine (PEI) to minimize nonspecific binding. Samples were transferred to the filterplates, washed with cold assay buffer, and dried overnight. Each well was counted in Microscint 0™ (Perkin Elmer) for 1 minute in triplicate.

O'Hayre M., Eichel K., Avino S., Zhao X., Steffen D.J., Feng X., Kawakami K., Aoki J., Messer K., Sunahara R., Inoue A., von Zastrow M, & Gutkind J.S. (2017). Genetic evidence that β-arrestins are dispensable for the initiation of β2-adrenergic receptor signaling to ERK. Science signaling, 10(484), eaal3395.

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Cell Proliferation Assay for Anticancer Drugs

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Example 5

Cell Proliferation Assay

Cancer cells were cultured in 96-well white plates (Perkin Elmer). They were synchronized for 24 hours in serum-deprived medium. After incubation of cells with unconjugated drugs (docetaxel, cabazitaxel, doxorubicin) or with drug-Katana peptide conjugates for 2 or 3 days, all media was aspirated and cells were pulse-labeled for 4 hours at 37° C./95% O₂/5% CO₂with media containing 2.5 μCi/mL [methyl-³H] thymidine (Perkin Elmer). Cells were washed, fixed, and dried before addition of scintillation fluid (Microscint 0, Perkin Elmer). After 24 hours, cell-associated tritium was quantified by counting on a plate reader (TopCount, Perkin Elmer). Incorporated [³H] thymidine was plotted for each drug concentration.

US11780882B2. Peptide compounds and peptide conjugates for the treatment of cancer through receptor-mediated chemotherapy (2023-10-10). TRANSFERT PLUS, S.E.C. [CA]. Inventors: Richard Béliveau [CA], Borhane Annabi [CA], Michel Demeule [CA], Alain Larocque [CA], Jean-Christophe Currie [CA], Cyndia Charfi [CA].

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Allosteric Modulator Binding Assay

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Cell membranes from FreeStyle 293 cells transiently expressing human M₁R were incubated with T‐495 or MK‐7622 (0.1‐30 μmol/L), ACh (0.003‐3000 μmol/L), and 4 nmol/L [³H]‐pirenzepine (PerkinElmer) in assay buffer (20 mmol/L HEPES, 100 mmol/L NaCl, 10 mmol/L MgCl₂, and 0.1% fatty acid free BSA) for 2 hours at room temperature. The binding was terminated by filtration through GF/C filter plates (PerkinElmer) using a cell harvester (PerkinElmer) and five washed with 300 μL of 50 mmol/L Tris‐HCl. The GF/C plates were dried at 42°C; then, 25 μL of microscint 0 (PerkinElmer) was added. Radioactivity was counted using Topcount (PerkinElmer). Nonspecific binding was defined in the presence of 10 μmol/L atropine. To calculate the cooperativity of a PAM, the [³H]‐pirenzepine binding assay data were fitted to the allosteric ternary complex model,35 using GraphPad Prism 5 software:

Y = \frac{\frac{[C]}{K_{C}} + \frac{α_{BC} [B] [C]}{K_{B} K_{C}}}{1 + \frac{[A]}{K_{A}} + \frac{[B]}{K_{B}} + \frac{[C]}{K_{C}} + \frac{α_{AB} [A] [B]}{K_{A} K_{B}} + \frac{α_{BC} [B] [C]}{K_{B} K_{C}}}

where Y is the fractional specific [³H]‐pirenzepine binding; [A], [B], and [C] are the concentrations of ACh, a PAM, and [³H]‐pirenzepine, respectively; K_A, K_B, and K_C are the equilibrium dissociation constants of ACh, a PAM, and [³H]‐pirenzepine, respectively; and α_AB and α_BC are the cooperativities between a PAM and ACh or [³H]‐pirenzepine, respectively.

Mandai T., Sako Y., Kurimoto E., Shimizu Y., Nakamura M., Fushimi M., Maeda R., Miyamoto M, & Kimura H. (2020). T‐495, a novel low cooperative M1 receptor positive allosteric modulator, improves memory deficits associated with cholinergic dysfunction and is characterized by low gastrointestinal side effect risk. Pharmacology Research & Perspectives, 8(1), e00560.

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Radioligand Binding Assay for NTS Receptor

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¹²⁵I-NTS Binding was assessed in microconic tubes in a 400 µL volume: 30 µg of cell membranes were mixed in Tris–HCl (50 mM, pH 7,4) supplemented with BSA (0.2%) with a concentration range of ¹²⁵I-NTS (0 to 4 nM) and left to incubate for 60 min. To assess the non-specific¹²⁵I-NTS binding the same binding mix was supplemented with cold NTS (10 µM). The binding reactions were terminated by filtration onto GF/C filter plate (PerkinElmer, 6005174) with a harvester filtration system (Filtermate Harvester Packard, PerkinElmer), followed by washes with Tris–HCL (50 mM, pH 7.4) and allowed to dry. MicroScint-0 (20 µL, PerkinElmer, 6013611) was added to each well and ¹²⁵I radioactivity was measured on TopCount (Packard, PerkinElmer, NXT). The Bmax is calculated as follow M = count in cpm / 10-9/2200 Ci/mmol/0.6/2.2/400 µL/decay factor then calculated Bmax value in fmol/mg = concentration in M*0.0004*10¹⁵/mg cell membrane proteins.

Libanje F., Delille R., Young P.A., Rolland S., Meyer-Losic F., Lewkowicz E, & Klinz S. (2023). NTSR1 glycosylation and MMP dependent cleavage generate three distinct forms of the protein. Scientific Reports, 13, 4663.

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Rac Expression Impact on P. aeruginosa

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The impact of Rac expression on P. aeruginosa PAO1 transcription was investigated using tritium-labelled uridine precursors, as described previously [31 (link)]. Briefly, an exponentially growing culture (OD₆₀₀ of 0.3) was labeled for 10 min with 1 μCi/mL (5,6-³H)-uridine (PerkinElmer, Waltham, MA, USA). Samples were taken 0, 10, 30 and 60 min after the induction of Rac with 1 mM IPTG and precipitated in 5% ice-cold trichloroacetic acid (TCA). The precipitate was transferred to a Unifilter-96 GF/C (PerkinElmer) using a Filtermate 96 Harvester (Packard) and washed. After the addition of MicroScint 0 (PerkinElmer), the radioactive signal was measured with a TopCount NXT Microplate Scintillation Counter (PerkinElmer). The experiment was repeated on three independent occasions. Non-induced cultures were grown in parallel to permit normalization for each timepoint.

Ceyssens P.J., De Smet J., Wagemans J., Akulenko N., Klimuk E., Hedge S., Voet M., Hendrix H., Paeshuyse J., Landuyt B., Xu H., Blanchard J., Severinov K, & Lavigne R. (2020). The Phage-Encoded N-Acetyltransferase Rac Mediates Inactivation of Pseudomonas aeruginosa Transcription by Cleavage of the RNA Polymerase Alpha Subunit. Viruses, 12(9), 976.

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Radioligand Binding Assay for Dopamine Receptor

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Membranes were prepared from Sf9 cells co-infected with Gα_i1β₁γ₂ and wild-type human DRD2_long (DRD2^WT) as described^{31 (link)}. Membranes (7–10 μg) were pretreated with or without antibody fragment scFv16 (1 μg/10 μg membranes) for 30 min at room temp and then incubated for 2 hr at 30^oC in binding buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM ascorbic acid, 0.001% BSA, 100 nM GDP) and 0.03 – 1.6 nM [³H]spiperone (for saturations) or with 0.13 nM [³H]spiperone (for competitions) along with varying concentrations of dopamine (or with 20 μM (+)butaclamol to determine non-specific binding). Samples were subject to rapid filtration through GF/B Unifilter™ plates (Packard) and rinsed with ice cold binding buffer to remove free [³H]spiperone. Filter plates were dried before adding Microscint 0™ (Perkin Elmer) and counting bound [³H]spiperone using a TopCount™ (Perkin Elmer). All data were analyzed using Prism (Graphpad Software Inc, San Diego CA).

Yin J., Chen K.Y., Clark M.J., Hijazi M., Kumari P., Bai X.C., Sunahara R.K., Barth P, & Rosenbaum D.M. (2020). Structure of a D2 dopamine receptor-G protein complex in a lipid membrane. Nature, 584(7819), 125-129.

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Cell Proliferation Assay for Cancer Cells

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Example 5

Cell Proliferation Assay

Cancer cells were cultured in 96-well white plates (Perkin Elmer). They were synchronized for 24 hours in serum-deprived medium. After incubation of cells with unconjugated drugs (docetaxel, cabazitaxel, doxorubicin) or with drug-Katana peptide conjugates for 2 or 3 days, all media was aspirated and cells were pulse-labeled for 4 hours at 37° C./95%/O₂/5%/CO₂with media containing 2.5 ρCi/mL [methyl-³H] thymidine (Perkin Elmer). Cells were washed, fixed, and dried before addition of scintillation fluid (Microscint 0, Perkin Elmer). After 24 hours, cell-associated tritium was quantified by counting on a plate reader (TopCount, Perkin Elmer). Incorporated [³H] thymidine was plotted for each drug concentration.

US11034727B2. Peptide compounds and peptide conjugates for the treatment of cancer through receptor-mediated chemotherapy (2021-06-15). TRANSFERT PLUS, S.E.C. [CA]. Inventors: Richard Béliveau [CA], Borhane Annabi [CA], Michel Demeule [CA], Alain Larocque [CA], Jean-Christophe Currie [CA], Cyndia Charfi [CA].

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Radioligand Binding Assay for Dopamine Receptor

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