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Cytoscint

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Cytoscint is a scintillation cocktail designed for liquid scintillation counting. It is a non-hazardous, biodegradable, and non-flammable solution that can be used to measure radioactive samples.

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Lab products found in correlation

Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Ls6000ta scintillator, by Beckman Coulter (2 mentions) 14c palmitate, by PerkinElmer (2 mentions) 14c glucose, by PerkinElmer (2 mentions) Fugene 6, by Promega (2 mentions) Ls 6000sc scintillation counter, by Beckman Coulter (1 mentions) L 3 4 3h glutamic acid, by PerkinElmer (1 mentions) T4 pnk, by Thermo Fisher Scientific (1 mentions) Illustra microspin g 25 column, by GE Healthcare (1 mentions) γ 32p atp, by PerkinElmer (1 mentions) Gf c filter, by Cytiva (1 mentions) Tri carb liquid scintillation analyzer, by Hewlett-Packard (1 mentions) 1 14c palmitate, by PerkinElmer (1 mentions) Benzethonium hydroxide, by Merck Group (1 mentions)

10 protocols using cytoscint

1

Measuring Skeletal Muscle Fatty Acid Oxidation

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Soleus and gastrocnemius muscle of overnight-fasted mice were assayed as described (28 (link)) with modifications (29 (link)). Muscle was homogenized in 10 mmol/L Tris (pH 7.2), 300 mmol/L sucrose, and 2 mmol/L EDTA, injected by syringe into a sealed beaker to be incubated at 30°C for 45 min in the presence of 0.2 mmol/L of [1-14C]palmitate (0.5 µCi/mL) and 2 mmol/L ATP in incubation buffer (100 mmol/L sucrose, 10 mmol/L Tris-HCl, 5 mmol/L potassium phosphate, 80 mmol/L KCl, 1 mmol/L MgCl2, 2 mmol/L l-carnitine, 0.1 mmol/L malic acid, 0.05 mmol/L CoA, 1 mmol/L dithiothreitol, 0.2 mmol/L EDTA, and 0.5% BSA, pH 7.4). The reaction was terminated with glacial acetic acid, and trapped CO2 radioactivity was measured by liquid scintillation in CytoScint (MP Biomedicals).
Al-Share Q.Y., DeAngelis A.M., Lester S.G., Bowman T.A., Ramakrishnan S.K., Abdallah S.L., Russo L., Patel P.R., Kaw M.K., Raphael C.K., Kim A.J., Heinrich G., Lee A.D., Kim J.K., Kulkarni R.N., Philbrick W.M, & Najjar S.M. (2015). Forced Hepatic Overexpression of CEACAM1 Curtails Diet-Induced Insulin Resistance. Diabetes, 64(8), 2780-2790.
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2

Glutamate Uptake Driven by TF0F1

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To measure uptake driven by TF0F1, 25 µl proteoliposomes containing 2–3.5 µg VGLUT were diluted into 775 µl uptake buffer (150 mM K gluconate, 5 mM HEPES-KOH pH 7.3, 5 mM Mg gluconate, 0.2 mM glutamate, 10 µCi/ml L-[3,4-3H]-glutamic acid (PerkinElmer) and 2 mM MgATP), for a final Cl− concentration of 5 mM. To measure uptake in the absence of TF0F1 (VGLUT only), the uptake buffer at pH 7.3 contained 150 mM K gluconate, 10 mM HEPES-KOH pH 7.3 and 5 mM Mg gluconate (KGluc pH 7.3). At pH 6.0, the buffers contained 0.2 mM glutamate and 10 µCi/ml L-[3,4-3H]-glutamic acid with either 150 mM K gluconate, 10 mM MES-KOH pH 6.0 and 5 mM Mg gluconate (KGluc pH 6) or 160 mM KCl, 10 mM MES-KOH pH 6.0 and 5 mM MgCl2 (KCl pH 6). The reactions were incubated at 30° C for the times indicated and stopped by rapid filtration through 0.45 µm HAWP discs (Millipore). The filters were washed 3 times with cold wash buffer (150 mM K gluconate, 5 mM HEPES-KOH, pH 7.3, 5 mM Mg gluconate, dried, solubilized in CytoScint (MP Biomedicals) and the bound radioactivity measured using an LS 6000SC scintillation counter (Beckman). All experiments were performed in duplicate or triplicate and repeated at least three times. Glutamate uptake was normalized to the mass of reconstituted VGLUT added to each reaction.
Eriksen J., Chang R., McGregor M., Silm K., Suzuki T, & Edwards R.H. (2016). Protons Regulate Vesicular Glutamate Transporters through an Allosteric Mechanism. Neuron, 90(4), 768-780.
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3

Radiolabeling and Binding of Poly I:C to PKR

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One microgram of reconstituted poly I:C was labelled using 10 units of T4 polynucleotide kinase (T4 PNK, Thermo Fisher Scientific) for 10 min at 37°C in a 25-μl reaction containing 25 μCi [γ-32P]-ATP (Perkin Elmer). The reaction was terminated by adding 5 mM EDTA to the reaction mixture and the labeled poly I:C was purified using an illustraMicrospin G-25 column (GE Healthcare, Marlborough, MA). In a pull-down assay, HeLa cells in a 10-cm culture dish were transfected with a Myc-Flag-tagged PKR expression vector or an empty vector. The Myc-Flag-tagged PKR protein in the HeLa cell lysate was immunoprecipitated using 80 μl of anti-mycEZview beads (Sigma-Aldrich) overnight at 4°C. The beads were then washed four times with IP buffer and subsequently resuspended in 100 μl of IP buffer. Approximately, 40 μl of the resuspended beads coated with PKR were incubated with a mixture of 100 ng [γ-32P]-labelled poly I:C and 500 ng of purified recombinant ORF57-Flag protein or BSA (negative control) in 750 μl of IP buffer for 2 h at room temperature. The beads were extensively washed with IP buffer, resuspended in 5 ml of liquid scintillation cocktail (CytoScint, MP Biochemicals, Santa Ana, CA) and radioactivity was counted by a liquid scintillation counter.
Sharma N.R., Majerciak V., Kruhlak M.J, & Zheng Z.M. (2017). KSHV inhibits stress granule formation by viral ORF57 blocking PKR activation. PLoS Pathogens, 13(10), e1006677.
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4

Binding Assay for [3H]PK 11195

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Binding assays of [3H]PK 11195, including displacement studies were done as previously described.76 (link) The reaction mixtures for the binding assays contained 400 μl of homogenized rat kidney membranes (5 mg of homogenate/ml) and 25 μl of [3H]PK 11195 (2 nM final concentration)76 (link) in the absence (total binding) or presence of various concentrations (10-10 M–10-5 M) of the synthesized compounds described in this study. After incubation for 60 min at 4 °C, samples were filtered under vacuum over Whatman GF/C filters and washed three times with 5 ml of 50 mM Tris-HCl buffer, pH 7.4. The filters were incubated in CytoScint (MP Biomedicals) and radioactivity was measured after 12 h with a 1600 CA Tri-Carb liquid scintillation analyzer (Packard). Inhibitory constant (Ki) values were calculated by the equation Ki=IC50/(1+C/Kd), where C=[3H] PK 11195 concentration, IC50=concentration causing 50% inhibition of [3H]PK 11195 binding and Kd=2 nM (from Scatchard analysis of [3H]PK 11195 binding to kidney membranes).
Vainshtein A., Veenman L., Shterenberg A., Singh S., Masarwa A., Dutta B., Island B., Tsoglin E., Levin E., Leschiner S., Maniv I., Pe’er L., Otradnov I., Zubedat S., Aga-Mizrachi S., Weizman A., Avital A., Marek I, & Gavish M. (2015). Quinazoline-based tricyclic compounds that regulate programmed cell death, induce neuronal differentiation, and are curative in animal models for excitotoxicity and hereditary brain disease. Cell Death Discovery, 1, 15027-.
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5

Radioligand Binding Assay for GPCR

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Binding curves were obtained by incubating the DDM purified wild-type, bimane- and Atto655-labeled receptors in the presence of M1 FLAG–sepharose and 2 mM Ca2+, under shaking at room temperature for 1.5h. The antagonist [3H]-dihydroalprenolol (DHA) (PerkinElmer) was used to obtain saturation binding curves. Competition binding measurements for salmeterol and epinephrine were performed in a similar way, using DHA at a concentration slightly above Kd, as determined by saturation binding. Non-specific binding was accounted for by measuring in the presence of 10μM cold alprenolol. Beads were harvested using a 48-well Brandell harvester and counted in a LS6000TA scintillator (Beckman) using Cytoscint (MP Biomedical).
Masureel M., Zou Y., Picard L.P., van der Westhuizen E., Mahoney J.P., Rodrigues J.P., Mildorf T.J., Dror R.O., Shaw D.E., Bouvier M., Pardon E., Steyaert J., Sunahara R.K., Weis W.I., Zhang C, & Kobilka B.K. (2018). Structural insights into binding specificity, efficacy and bias of a β2AR partial agonist. Nature chemical biology, 14(11), 1059-1066.
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6

Radioligand Binding Assay for GPCR

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Binding curves were obtained by incubating the DDM purified wild-type, bimane- and Atto655-labeled receptors in the presence of M1 FLAG–sepharose and 2 mM Ca2+, under shaking at room temperature for 1.5h. The antagonist [3H]-dihydroalprenolol (DHA) (PerkinElmer) was used to obtain saturation binding curves. Competition binding measurements for salmeterol and epinephrine were performed in a similar way, using DHA at a concentration slightly above Kd, as determined by saturation binding. Non-specific binding was accounted for by measuring in the presence of 10μM cold alprenolol. Beads were harvested using a 48-well Brandell harvester and counted in a LS6000TA scintillator (Beckman) using Cytoscint (MP Biomedical).
Masureel M., Zou Y., Picard L.P., van der Westhuizen E., Mahoney J.P., Rodrigues J.P., Mildorf T.J., Dror R.O., Shaw D.E., Bouvier M., Pardon E., Steyaert J., Sunahara R.K., Weis W.I., Zhang C, & Kobilka B.K. (2018). Structural insights into binding specificity, efficacy and bias of a β2AR partial agonist. Nature chemical biology, 14(11), 1059-1066.
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7

Measuring Cardiac Fatty Acid Oxidation

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FA oxidation was measured using [1-14C]-palmitate in heart tissue and isolated cardiomyocytes as previously described.21 (link) The heart tissue samples were incubated in modified Krebs–Henseleit buffer containing 1.5% FA-free bovine serum albumin, 5 mmol l–1 glucose, 1 mmol l–1 palmitate and 0.5 μCi ml–1 [14C]palmitate (Perkin Elmer, Courtaboeuf, France) for 60 min. Isolated cardiomyocytes were incubated in the same modified Krebs–Henseleit buffer, but without glucose. After incubation, tissues were homogenized in 800 μl lysis buffer. Complete oxidation was determined by acidifying the incubation buffer with 1 ml of 1 mol l–1 H2SO4, and the 14CO2 was trapped by benzethonium hydroxide (Sigma) placed in a 0.5 ml microtube. After 120 min, the radioactivity was counted (Cytoscint; MP Biomedicals, Strasbourg, France). Similarly, glucose oxidation was assessed by measuring the [14C]-glucose oxidation in cardiac tissue as previously described. Sample incubation for 1 h was performed using a modified Krebs–Henseleit buffer containing 0.2% bovine serum albumin, 20 mM Hepes, 10 mM glucose, 0.8 μCi ml–1 [14C]glucose (Perkin Elmer). Results were normalized for mg of proteins.
Alfarano C., Foussal C., Lairez O., Calise D., Attané C., Anesia R., Daviaud D., Wanecq E., Parini A., Valet P, & Kunduzova O. (2014). Transition from metabolic adaptation to maladaptation of the heart in obesity: role of apelin. International Journal of Obesity (2005), 39(2), 312-320.
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8

Quantifying Androgen Receptor Activity in HEK293 Cells

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HEK293 cells were transfected with vectors expressing wt-AR or AR mutants using FuGene6 (Promega) and 100,000 cells were plated in a 24-well plate (DMEM, 8% CFS) coated with 0.2% gelatin. Following overnight incubation, cells were treated with ligand in the presence of 0.1 nM 3H-R1881. To determine background levels of radioactivity, control wells were treated with 500X cold R1881 (50 nM). After 2 hr incubation, cells were lysed using 200 μl lysis buffer (2% SDS, 10% Glycerol, 10 mM Tris-HCl [pH 6.8]); then volumes were increased to 500 μl using 10 mM Tris-HCl (pH 8.0). 300 μl of the lysates were added to 3 mL of Cytoscint (MP Biomedicals) and analyzed by scintillation counting (Beckman LS 6000SC). Lysate protein levels were quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific) per the manufacturer’s instructions.
Pollock J.A., Wardell S.E., Parent A.A., Stagg D.B., Ellison S.J., Alley H.M., Chao C.A., Lawrence S.A., Stice J.P., Spasojevic I., Baker J.G., Kim S.H., McDonnell D.P., Katzenellenbogen J.A, & Norris J.D. (2016). Inhibiting androgen receptor nuclear entry in castration-resistant prostate cancer. Nature chemical biology, 12(10), 795-801.
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9

Measuring Fatty Acid and Glucose Oxidation in Cardiomyocytes

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The oxidation of FA was measured using [1–14C]-palmitate in heart tissue and isolated cardiomyocytes as previously described [26 (link)]. The heart tissue samples were incubated in modified Krebs–Henseleit buffer containing 1.5% FA-free bovine serum albumin, 5 mmol l–1 glucose, 1 mmol l–1 palmitate and 0.5 μCi ml–1 [14C]palmitate (PerkinElmer, Courtaboeuf, France) for 60 min. Isolated cardiomyocytes were incubated in the same modified Krebs–Henseleit buffer, but without glucose. After incubation, tissues were homogenized in 800 μl lysis buffer. Complete oxidation was determined by acidifying the incubation buffer with 1 ml of 1 mol l–1 H2SO4, and the 14CO2 was trapped by benzethonium hydroxide (Sigma) placed in a 0.5 ml microtube. After 120 min, the radioactivity was counted (Cytoscint; MP Biomedicals, Strasbourg, France). Similarly, glucose oxidation was assessed by measuring the [14C]-glucose oxidation in cardiac tissue as previously described. Sample incubation for 1 h was performed using a modified Krebs–Henseleit buffer containing 0.2% bovine serum albumin, 20 mM Hepes, 10 mM glucose, 0.8 μCi ml–1 [14C]glucose (PerkinElmer). Results were normalized for mg of proteins.
Martinelli I., Timotin A., Moreno-Corchado P., Marsal D., Kramar S., Loy H., Joffre C., Boal F., Tronchere H, & Kunduzova O. (2021). Galanin promotes autophagy and alleviates apoptosis in the hypertrophied heart through FoxO1 pathway. Redox Biology, 40, 101866.
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10

Quantifying Androgen Receptor Activity in HEK293 Cells

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HEK293 cells were transfected with vectors expressing wt-AR or AR mutants using FuGene6 (Promega) and 100,000 cells were plated in a 24-well plate (DMEM, 8% CFS) coated with 0.2% gelatin. Following overnight incubation, cells were treated with ligand in the presence of 0.1 nM 3H-R1881. To determine background levels of radioactivity, control wells were treated with 500X cold R1881 (50 nM). After 2 hr incubation, cells were lysed using 200 μl lysis buffer (2% SDS, 10% Glycerol, 10 mM Tris-HCl [pH 6.8]); then volumes were increased to 500 μl using 10 mM Tris-HCl (pH 8.0). 300 μl of the lysates were added to 3 mL of Cytoscint (MP Biomedicals) and analyzed by scintillation counting (Beckman LS 6000SC). Lysate protein levels were quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific) per the manufacturer’s instructions.
Pollock J.A., Wardell S.E., Parent A.A., Stagg D.B., Ellison S.J., Alley H.M., Chao C.A., Lawrence S.A., Stice J.P., Spasojevic I., Baker J.G., Kim S.H., McDonnell D.P., Katzenellenbogen J.A, & Norris J.D. (2016). Inhibiting androgen receptor nuclear entry in castration-resistant prostate cancer. Nature chemical biology, 12(10), 795-801.
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