Ecoscint h

Manufactured by National Diagnostics

Sourced in United States

Ecoscint H is a scintillation cocktail solution used for liquid scintillation counting. It is designed to be compatible with a wide range of sample types, including aqueous, organic, and biological samples.

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Lab products found in correlation

14c aa, by PerkinElmer (1 mentions) 3h uridine, by PerkinElmer (1 mentions) 3h thymidine, by PerkinElmer (1 mentions) Ls6500, by Beckman Coulter (1 mentions) 35s methionine, by PerkinElmer (1 mentions) Perchloric acid, by Thermo Fisher Scientific (1 mentions) Carnitine, by Merck Group (1 mentions) 1 14c palmitate, by American Radiolabeled Chemicals (1 mentions) Ls 6000 ic liquid scintillation counter, by Beckman Coulter (1 mentions) Gbr12909, by Merck Group (1 mentions) 2 deoxy d 1 3h glucose, by PerkinElmer (1 mentions) Liquid scintillation counter, by Beckman Coulter (1 mentions) 14c palmitate, by American Radiolabeled Chemicals (1 mentions) 3h corticosterone, by PerkinElmer (1 mentions) Microbeta2, by PerkinElmer (1 mentions)

10 protocols using ecoscint h

Quantifying Prostanoid Levels in Mice

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Prostanoid levels were determined in mouse tissue extracts from 0 (non-infected) and 21 d.p.i. by Metabolon Inc., and expressed as ScaledImpData as previously described [40 (link)]. To determine in vitro prostanoid production, heart infiltrating cells were magnetically sorted as described above and incubated 30 minutes at 37°C in 500 μl of RPMI without Fetal bovine serum (FBS) in the presence of 25 μM [¹⁴C] AA, PerkinElmer (Massachusetts, USA). 500 μl of 2% acetic acid in cold methanol was added to extract and preserve AA derivatives. Samples were vortexed and the air inside the tube was substituted by inert nitrogen gas. Samples were kept frozen at -80°C until HPLC was performed. HPLC device was composed by a Beckman Solvent Module 126 with the column Ultraphere ODS (C-18, Beckman-Coulter) 5 μm diameter sphere particle, 4.5 mm and 25 cm column diameter and length respectively and a Beckman 171 Radioisotope Detector. Scintillation liquid Ecoscint H was purchased from National Diagnostics. Prostanoids were resolved with the isocratic flow (1ml/min) of the mobile phase: Acetonitrile/water/acetic acid 33:67:0.1 v/v/v. Standards were produced using [¹⁴C] Arachidonic Acid and different cell types expressing the respective enzymes, and [¹⁴C] Arachidonic Acid incubated in medium was used as input control as described [41 (link)].

Guerrero N.A., Camacho M., Vila L., Íñiguez M.A., Chillón-Marinas C., Cuervo H., Poveda C., Fresno M, & Gironès N. (2015). Cyclooxygenase-2 and Prostaglandin E2 Signaling through Prostaglandin Receptor EP-2 Favor the Development of Myocarditis during Acute Trypanosoma cruzi Infection. PLoS Neglected Tropical Diseases, 9(8), e0004025.

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Measurement of Macromolecular Synthesis Rates

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Cells were grown as described above and treated with OFX (5 μg/ml) at an OD_600nm of 0.3. One milliliter of culture samples was collected at indicated time points and incubated with [³H]-thymidine (1 μCi/ml), [³H]-uridine (1 μCi/ml), or [³⁵S]-methionine (3 μCi/ml; PerkinElmer) for 5 min. Untreated samples were collected before the addition of OFX. Samples were precipitated overnight at 4°C with 5 ml of cold 10% trichloroacetic acid (TCA). The next day, samples were filtered on 0.45-μm nitrocellulose and washed with 15 ml of 10% TCA. Filter-retained material was counted in 10 ml of scintillation liquid (Ecoscint H, National Diagnostics, LS-275) in a liquid scintillation counter (Beckman LS 6500). The integration time per read was 2 min. Background signals from cells without radioactivity were subtracted. The incorporation rate is the ratio of the CPM counted at each time point to the corresponding zero time. Controls for DNA, RNA, and protein synthesis arrest consist of cultures treated with MMC (10 μg/ml), rifampicin (100 μg/ml), and Cm (30 μg/ml), respectively.

Cayron J., Oms T., Schlechtweg T., Zedek S, & Van Melderen L. (2024). TisB protein is the single molecular determinant underlying multiple downstream effects of ofloxacin in Escherichia coli. Science Advances, 10(13), eadk1577.

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Dopamine Uptake Assay in Flp-DAT Cells

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Flp-DAT cells were counted and 1 × 10⁶ cells were exposed to different concentrations of DA where 1% of the total concentration consisted of [³H]DA. The uptake reaction was performed for 10 min at 37°C in an external solution containing (in mM): 130 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, 10 glucose, pH adjusted to 7.4. Non-specific uptake was determined adding 10 μM methylenedioxypyrovalerone (MDPV, a potent hDAT blocker) [31 (link), 32 (link)]. After the incubation period, cells were centrifuged, washed once with PBS, centrifuged again and the cell pellets were resuspended in Ecoscint H (National Diagnostics, Atlanta, GA, USA); radioactivity was measured in a liquid scintillation counter.
Dose response-experiments were fit to the following expression:

Y (x) = \frac{Y_{max}}{1 + 10 exp [{log {E C}_{50} - log x} * n]}

Where x is the concentration of the tested compound, Y(x) is the response measured, Y_max is the maximal response, EC₅₀ is the concentration that yields half-maximal response, and n is the Hill slope parameter. Competition assays were carried out adding a variable concentration of cold DA or cold S(+)AMPH to a constant 10 μM DA solution containing 1% [³H]DA. The inhibition constant (K_i) was estimated using the Cheng-Prusoff equation.

Cameron K.N., Solis E J.r., Ruchala I., De Felice L.J, & Eltit J.M. (2015). Amphetamine activates calcium channels through dopamine transporter-mediated depolarization. Cell calcium, 58(5), 457-466.

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Measuring Fatty Acid Oxidation in Brown Adipocytes

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Rates of fatty acid oxidation were determined in primary brown adipocytes according to the conversion of [1–¹⁴C] palmitate (55 mCi/mmol: American Radiolabeled Chemicals; St. Louis, MO, USA) into ¹⁴C-acid soluble metabolites (ASM) and -CO₂[18] (link). Briefly, matured brown adipocytes were incubated for 6 h in 2 ml of fresh culture medium with 10% FBS supplemented with 1 mM carnitine (Sigma–Aldrich) and 0.25 μCi [1–¹⁴C] palmitate/ml plus 200 μM palmitate conjugated with fatty acid-free BSA. To determine ASM, the culture medium was mixed in conical tubes with 200 μl of 70% perchloric acid (Fisher Scientific; Pittsburgh, PA, USA) with filter papers soaked in 2N NaOH placed in the caps in order to trap ¹⁴C–CO₂. After incubation for 1 h on a horizontal shaker, the filter papers were removed, and the acidified medium was incubated overnight at 4 °C and then centrifuged at 14,000 × g for 20 min ¹⁴C-labeled CO₂ in the filter paper and ASM in the supernatant were dissolved in Ecoscint H (National Diagnostics; Atlanta, GA, USA) and were counted using a LS6000IC liquid scintillation counter (Beckman Coulter; Danvers, MA).

Okada K., LeClair K.B., Zhang Y., Li Y., Ozdemir C., Krisko T.I., Hagen S.J., Betensky R.A., Banks A.S, & Cohen D.E. (2016). Thioesterase superfamily member 1 suppresses cold thermogenesis by limiting the oxidation of lipid droplet-derived fatty acids in brown adipose tissue. Molecular Metabolism, 5(5), 340-351.

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Dopamine Uptake Assay in Brain Slices

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The slices were obtained as described above and allowed to recover (⩾1 h at 28 °C) with continuous oxygenation. Following the recovery, the slices were treated with the drug (that is, GLP-1) for 10 min followed by the application of 50 nm [³H]DA for 10 min with the drug still present at 28 °C. When Ex-9 was used, the slices were pretreated with Ex-9 for 10 min before the addition of drug/vehicle. Total protein was taken, and the samples were processed for protein concentration using a Bio-Rad (Philadelphia, PA, USA) protein assay and spectrometry at 595 nm. Equivalent volumes of the sample were added to 3 ml of scintillation fluid (Ecoscint H, National Diagnostics, Atlanta, GA, USA) and radioactive counts were measured by scintillation counter. [³H]DA was divided by protein and normalized to average control uptake from paired vehicle-treated slices from the same animal. The uptake protocol and DAT specificity were validated in slices treated with DAT blocker GBR-12909 (Sigma Aldrich, St. Louis, MO, USA), which blocked uptake in LS by 58.2% (t(5)=11.97; P<0.0001 by Student's t-test; n=6).

Reddy I.A., Pino J.A., Weikop P., Osses N., Sørensen G., Bering T., Valle C., Bluett R.J., Erreger K., Wortwein G., Reyes J.G., Graham D., Stanwood G.D., Hackett T.A., Patel S., Fink-Jensen A., Torres G.E, & Galli A. (2016). Glucagon-like peptide 1 receptor activation regulates cocaine actions and dopamine homeostasis in the lateral septum by decreasing arachidonic acid levels. Translational Psychiatry, 6(5), e809-.

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2-Deoxy-D-[1-3H]Glucose Uptake Assay

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Cells (1 × 10⁶ cells) were grown in six-well plates and incubated for 24 h. Cells were incubated with 1 μCi (1 μl) of 2-deoxy-d-[1-³H]glucose (Perkin Elmer) in 1 ml of media for 20 min at 37°C. The cells were washed two times with ice-cold PBS, and lysed in 1 ml of 1% SDS. Cell lysate 0.8 ml in 4.5 ml Ecoscint H (National Diagnostics LS-275) was counted on a scintillation device for 1 min. All experiments were done in triplicates wells, and repeated at least two times.

Leung E.Y., Kim J.E., Askarian-Amiri M., Joseph W.R., McKeage M.J, & Baguley B.C. (2014). Hormone Resistance in Two MCF-7 Breast Cancer Cell Lines is Associated with Reduced mTOR Signaling, Decreased Glycolysis, and Increased Sensitivity to Cytotoxic Drugs. Frontiers in Oncology, 4, 221.

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Measuring Muscle Fatty Acid Oxidation

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Rates of fatty acid oxidation in muscle tissues were measured by degradation of ¹⁴C-palmitate (American Radiolabeled Chemicals; St. Louis, MO, USA) into ¹⁴C acid soluble metabolites (ASM) and ¹⁴C-labeled CO₂^{46 (link),48}. Briefly, gastrocnemius muscle strips were collected from 6 h fasted mice. Tissues were kept on ice no longer than 30 min. Muscle tissues were minced and homogenized in a Dounce homogenizer followed by centrifugation for 10 min at 420 × g. Supernatants were transferred to microtubes containing 0.4 μCi ¹⁴C-palmitate/500 μM palmitate conjugated with 0.7% fatty acid-free BSA and incubated at 37 °C for 30 min. ¹⁴C-labeled CO₂ produced by TCA cycle was captured onto filter paper soaked with 1 M NaOH and ¹⁴C-labeled ASM were separated with 1 M perchloric acid. ¹⁴C-labeled CO₂ in the filter paper and ¹⁴C-labeled ASM in the supernatant were dissolved in Ecoscint H (National Diagnostics; Atlanta, GA, USA) and were counted using a liquid scintillation counter (Beckman Coulter; Danvers, MA).

Druzak S.A., Tardelli M., Mays S.G., El Bejjani M., Mo X., Maner-Smith K.M., Bowen T., Cato M.L., Tillman M.C., Sugiyama A., Xie Y., Fu H., Cohen D.E, & Ortlund E.A. (2023). Ligand dependent interaction between PC-TP and PPARδ mitigates diet-induced hepatic steatosis in male mice. Nature Communications, 14, 2748.

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Serotonin Uptake Assay in Flp-In Cells

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Flp-In cells expressing hSERT were counted and 2×10⁶ cells were exposed to different concentrations of 5HT where 3% of a given concentration consisted of [³H]-5HT. The 5HT uptake was carried out for 10 min at 37°C in IS (see composition above). Non-specific uptake was measured adding 5 μM fluoxetine to the radioactive cocktail. Then the cells were centrifuged, washed once with cold 1X PBS, centrifuged again, cell pellets were resuspended in Ecoscint H (National Diagnostics, Atlanta, GA, USA), and the radioactivity counted using a liquid scintillation counter. The mean ± s.e.m. of 5 independent experiments done in duplicates are reported.

Ruchala I., Cabra V., Solis E J.r., Glennon R.A., De Felice L.J, & Eltit J.M. (2014). Electrical coupling between the human Serotonin Transporter and Voltage-Gated Ca2+ Channels. Cell calcium, 56(1), 25-33.

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Quantifying 11β-HSD Dehydrogenase Activity

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Small pieces of fresh epididymal adipose tissue were manually homogenized on ice, and homogenates were diluted to obtain a final concentration of 0.05 mg protein/mL. The reaction tubes containing 15 nM of mixed cold [3H]-corticosterone (Perkin Elmer) were covered and incubated at 37°C for 10 or 30 min. Steroids were extracted with ethyl acetate, and the dried pellet was resuspended in ethanol. Hormones were separated by thin layer chromatography in silica gel Polygram SIL N-HR/UV 254 (Macherey-Nagel, Düren, Germany) using diclormethane:acetone (4:1) as a mobile phase.
Cold standards of 11-dehydrocorticosterone and corticosterone were used to identify their specific zone migration. Each hormone zone was mixed with scintillation solution Ecoscint H (National Diagnostics, Nottingham, UK) in order to quantify the radioactivity. 11β-HSD dehydrogenase activity was expressed as pmol of 11β-dehydrocorticoterone produced per mg of protein and hour of incubation.

García-Eguren G., Giró O., Romero M.D.M., Grasa M, & Hanzu F.A. (2019). Chronic hypercortisolism causes more persistent visceral adiposity than HFD-induced obesity. The Journal of endocrinology, 242(2).

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Propionate Absorption Kinetics in Animals

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Anesthetized animals were either orally gavaged or rectally administered 1-14 C-labeled propionate (0.1 mCi/kg) (MP Biomedicals, Santa Ana, CA) mixed with sodium propionate (1 g/kg) and trace amounts of dextran blue (for visual confirmation of administration). Rectal administration was performed via 3-cm PE50 tubing attached to a blunt 23G needle. After administration, animals were sampled via tail vein bleeding for blood collection. At the end of the experiment, animals were euthanized via cervical dislocation, and digestive tract was removed for visual inspection of dextran blue. At all instances, orally administered bolus was confined to stomach and rectally administered bolus was confined between duodenum and distal colon. Radioactivity was measured by decolorizing 5 l of blood with hydrogen peroxide and mixing it with Ecoscint H (National Diagnostics, Atlanta, GA) at 1:10 (v/v) ratio and reading on a MicroBeta2 instrument (PerkinElmer, Waltham, MA).

Tirosh A., Calay E.S., Tuncman G., Claiborn K.C., Inouye K.E., Eguchi K., Alcala M., Rathaus M., Hollander K.S., Ron I., Livne R., Heianza Y., Qi L., Shai I., Garg R, & Hotamisligil G.S. (2019). The short-chain fatty acid propionate increases glucagon and FABP4 production, impairing insulin action in mice and humans. Science translational medicine, 11(489).

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