One milliliter of blood was taken at each time point from a peripheral vein into chilled 1.5-mL Eppendorf tubes coated with lithium fluoride, heparin, and 1 μg/50 μL EDTA. Collected samples were immediately centrifuged, plasma was aliquoted, and glucose concentrations were measured with a YSI 2700 autoanalyzer (Yellow Springs Instruments, Yellow Springs, OH). The rest of the plasma samples were stored at −20°F until ready for insulin measurements and [3-3H]glucose tracer assay in the case of the hyperinsulinemic-euglycemic clamp (EGC). A sandwich ELISA kit designed for porcine and canine insulin (80-INSPO-E01; ALPCO, Salem, NH) was used to assay the insulin. The ELISA has an identical sensitivity to porcine and canine insulin. The intra- and interassay coefficient of variance of insulin was 2.3 ± 0.3% and 2.9 ± 1.3%, respectively. Samples were processed on ice. Average baseline insulin measurements of each dog before the experiments were considered the fasting insulin level. The plasma samples from the EGC were processed according to Ader and Bergman (11 (link)), and the specific activity of the [3-3H]glucose tracer was assayed by a liquid scintillation counter (LS 6000SC; Beckman Coulter, Fullerton, CA).
Ls6000sc
Manufactured by Beckman Coulter
Sourced in United States
The LS6000SC is a laser-based particle size analyzer capable of measuring particle size distributions across a wide range of samples. It utilizes the principles of laser light scattering to determine the size of particles suspended in a liquid or gas.
Automatically generated - may contain errors
Lab products found in correlation
Mitochondria isolation kit, by Miltenyi Biotec (2 mentions)
Ysi 2700, by Yellow Springs Instruments (1 mentions)
Free glycerol reagent, by Merck Group (1 mentions)
Cytoscint liquid scintillation cocktail, by MP Biomedicals (1 mentions)
3h glycine, by PerkinElmer (1 mentions)
Ecoscint a, by National Diagnostics (1 mentions)
3h granisetron, by PerkinElmer (1 mentions)
13 protocols using ls6000sc
1
Plasma Insulin and Glucose Measurement
2
ER Binding Assay of Herbal Medicine
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HELN-ERα and HELN-ERβ cells were seeded into each well (7 × 104 cells/well) of a 96-well white opaque tissue culture plate with a clear bottom (Greiner reference 655098) and grown in the test culture medium for 24 hours in a 5% CO2 humidified atmosphere. At the end of the 24 hours, the cells were exposed to increasing dilutions of the TCM (1-0.0003%) in the presence of 0.3 nM [3H]-E2 (41.3 Ci/mmol specific activity) for 3 hours at 37°C. At the end of the incubation, the liquid in each well was aspirated and the cells were washed three times with 100 μl of cold phosphate buffered saline (PBS). Scintillation fluid (50 μl) (LS-6000-SC, Beckman-Coulter, Roissy, France) was added to each well, and the amount of [3H]-bound radioactivity in the mixture was determined using the MicroBeta Trilux luminometer (EGG Wallac, Turku, Finland). Non-specific binding was determined in the presence of 100 nM unlabeled E2. Specific binding was calculated by subtracting non-specific binding from total binding and was expressed as a percentage of the maximum ER binding (100%), which was obtained in absence of the herbal medicine. The IC50 value was defined as the dilution at which [3H]-E2 binding was 50%. Determinations were made in quadruplicate in three separate experiments. The absence of toxicity was determined by visual inspection.
3
Measuring Adipocyte Lipolysis and Lipid Uptake
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Assays were performed on explants of epididymal fat pads with intact ECM. Lipolysis was determined by glycerol release following 200 nmol/L isoproterenol treatment, using the Sigma free glycerol reagent, as described previously (24 (link)). For lipid uptake, fat pads were treated with 250 μmol/L tritiated oleic acid, with and without 10 nmol/L insulin. After incubation, fat pads were washed and homogenized in 0.1 mol/L NaOH, and radioactivity was determined using a Beckman LS6000SC scintillation counter (25 (link)).
4
Radioligand Displacement Assay for Serotonin Receptors
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The ability of BDS 391 to displace the binding of the radioligands [3H] 8-OH-DPAT (1.5 nM), [3H] Ketanserin (0.5 nM) or [3H] GR-65630 (0.69 nM), antagonists of, respectively, 5-HT1, 5-HT2 and 5-HT3 serotoninergic receptors, was determined by Ricerca Biosciences LCC, (Pharmacology Laboratories 158 Li-The Road, Peitou Taipei, Taiwan 112. Taiwan R.O.C.). Briefly, human recombinant CHO-K1 and HEK 293 cells membranes were incubated for 1 h at 25 °C, with 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 5 mM MgCl buffer containing the radioligands. Non-specific binding was determined using 10 µM Metergoline, 1 µM Mianserin or 10 µM MDL 72222, respectively. For the dissociation assays, BDS 391 was added to the incubation medium in concentrations up to 2 µM. Scintillation counting employing a Beckman LS6000SC (Fullerton, CA, USA) was used to determine radioactivity. For data (IC values) analysis, a nonlinear, least squares regression analysis was applied, using the MathIQä software (ID Business Solutions Ltd., Guildford, UK). Compound’s concentration able to inhibit radioligand binding by 50% of the maximum (IC50 value) was determined for each antagonist.
5
Cefadroxil Brain Distribution Measurement
6
Acetate Uptake in Metabolic Tissues
7
Mitochondrial Protein Synthesis Assay
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Mitochondria were isolated from BM Lin− cells using the Mitochondria Isolation kit (Miltenyi Biotech Inc) following manufacturer instructions. Cell-free mitochondrial translation was carried out using a modified protocol described previously (McKee et al., 2006 (link)). Briefly, mitochondria were incubated at 3 mg protein/ml in 30 μL protein synthesizing medium. The mitochondrial mix was incubated at 30 °C in the presence of vehicle (DMSO) or the mitochondrial translation inhibitor Chloramphenicol (CAP; 10 μM) for 5 min, and 4 μL (4 μCi) [3H]-leucine was then added to the mix and incubation continued for 60 min. The incorporation of [3H]-leucine into mitochondrial protein was determined by counting the [3H]-leucine-labeled proteins in a Beckman LS6000SC liquid scintillaton counter.
8
Intestinal Permeability Measurement via Mannitol
9
Kinetic Analysis of Glycyl-tRNA Synthetase
10
Radioligand Binding Assay in Transfected HEK 293 Cells
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Transfected HEK 293 cells were washed twice with phosphate buffered saline (PBS) at room temperature, scraped into 1 ml of ice-cold HEPES buffer (10 mM, pH 7.4), homogenised and frozen. After thawing, they were washed with HEPES buffer, resuspended, and 50 μg of cell suspension incubated in 0.5 ml HEPES buffer and the relevant concentration of radioligand at 0 °C. Non-specific binding was determined using 2 mM quipazine. Equilibrium reactions were incubated for at least 3 h for [3H] granisetron (63.5 Ci/mmol, PerkinElmer, Boston, Massachusetts, USA) and 48 h for [3H]VUF10166. Incubations were terminated by vacuum filtration onto GF/B filters pre-soaked in 0.3% polyethyleneimine, followed by three rapid washes with 3.5 ml ice cold buffer. Radioactivity was determined by scintillation in Ecoscint A (National Diagnostics, Atlanta, Georgia) using a Beckman LS6000SC (Fullerton, California, USA). Each method was performed on at least three independent cell samples on at least three separate days.
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