Skeletal muscle cells were cultured in 96-well CellBIND® microplates. After differentiation and conditioning, D-[14C(U)] glucose (18.5 kBq/mL, 200 µmol/L) or [1-14C] oleic acid (18.5 kBq/mL, 100 µmol/L) was added during 4 h of CO2 trapping [11 (link)]. CO2 production was measured in the presence or absence of 1 µmol/L of FCCP or 0.5 µmol/L of rotenone in combination with 2.5 µmol/L of antimycin A or 1 µmol/L of oligomycin. CO2 production and cell-associated radioactivity (CA) were assessed using a 2450 MicroBeta2 scintillation counter (PerkinElmer). The amount of radioactivity in the cells was related to the total cell protein content measured according to Bradford [12 (link)]. The sum of 14CO2 and CA was considered as the total substrate uptake.
2450 microbeta2 scintillation counter
Manufactured by PerkinElmer
Sourced in United States
The 2450 MicroBeta2 is a compact, high-performance scintillation counter designed for sensitive radioactivity detection and measurement. It utilizes advanced liquid scintillation technology to provide accurate and reliable results for a variety of applications.
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11 protocols using 2450 microbeta2 scintillation counter
1
Measuring Skeletal Muscle Metabolic Flux
2
Cellular Respiratory Substrate Uptake
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Cells were cultured on 96-well CellBIND® microplates. [1-14C]oleic acid (18.5 kBq/ml), 100 μM, or D-[14C(U)]glucose (21.46 kBq/ml), 200 μM, were given during 4 h CO2 trapping as previously described [22 (link)]. In brief, a 96-well UniFilter®-96 GF/B microplate was mounted on top of the CellBIND® culture plate and CO2 production was measured in DPBS medium with 10 mM HEPES and 1 mM L-carnitine adjusted to pH 7.2–7.3. CO2 production and cell-associated (CA) radioactivity were assessed using a 2450 MicroBeta2 scintillation counter (PerkinElmer). The sum of 14CO2 and CA radioactivity was taken as a measurement of total cellular uptake of substrate. Protein content in each well was measured according to Bradford [23 (link)].
3
A2B Adenosine Receptor Ligand Binding Assay
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Were performed using 1.5 nM [3H] PSB-603 and a competing unlabeled ligand at multiple concentrations diluted in assay buffer (50 mM Tris-HCl, 0.1% (w/v) CHAPS, pH 7.4). Binding was initiated by addition of CHO-spap-hA2BAR membrane aliquots to reach a total volume of 100 μL. Samples were incubated at 25 °C for 2 h to reach equilibrium. The incubation was terminated by rapid vacuum filtration over 96-well Whatman GF/C filter plates using a PerkinElmer Filtermate harvester (PerkinElmer, Groningen, Netherlands). Filters were subsequently washed ten times using ice-cold wash buffer (50 mM Tris-HCl, 0.1% (w/v) BSA, pH 7.4). Filter plates were dried at 55 °C for about 45 min and afterwards 25 μL Microscint (PerkinElmer) was added per well. Filter-bound radioactivity was determined by liquid scintillation spectrometry using a 2450 Microbeta2 scintillation counter (PerkinElmer).
4
Glucose and Fatty Acid Uptake Assay
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To measure glucose and FA accumulation, cells were cultured with 1 × 104 cells/well on Cytostar-T® 96 well scintillation microplate or 96-Scintiplate® (PerkinElmer), respectively, in DMEM-glu or DMEM-ole for 24 h. Thereafter, accumulation of D-[14C(U)]glucose (0.5 µCi/mL, 200 µM) or [1-14C]oleic acid (0.5 μCi/mL, 100 µM) was measured by scintillation proximity assay (SPA) over 24 h as previously described [33 (link)]. In brief, cells were incubated during 24 h in the presence of radiolabeled substrates with measurements at 0, 2, 4, 6, 8, and 24 h on a 2450 MicroBeta2 scintillation counter (PerkinElmer). The radiolabeled substrates taken up will accumulate in the adherent cells and become concentrated close to the scintillator embedded in the plastic bottom of each well. After the 24 h accumulation, the cells were harvested in 0.1 M NaOH and protein content in the lysates were measured by the Bio-Rad protein assay using a VICTOR™ X4 Multilabel Plate Reader (PerkinElmer).
5
Skeletal Muscle Substrate Oxidation
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Skeletal muscle cells (7000 cells/well) were cultured on 96-well CellBIND® microplates. Substrate, D-[14C(U)]glucose (18.5 kBq/ml, 200 μmol/l) or [1-14C]oleic acid (18.5 kBq/ml, 100 μmol/l) was given acutely during 4 h CO2 trapping as described previously (Wensaas et al., 2007 (link)). CO2 production (complete oxidation) and cell-associated radioactivity (CA) were assessed using a PerkinElmer 2450 MicroBeta2 scintillation counter (PerkinElmer). Protein content in each well was determined with the Bio-Rad protein assay using a VICTOR™ X4 Multilabel Plate Reader (PerkinElmer). Measurement of acid-soluble metabolites (ASM), reflecting incomplete fatty acid oxidation (β-oxidation) and mainly consists of tricarboxylic acid cycle metabolites, was performed as previously described by Skrede et al., (1994) (link) and modified by Bakke et al., (2012) . In short, 100 μl of the radiolabeled medium was transferred to an Eppendorf tube and precipitated with 300 μl cold HClO4 (1 mol/l) and 30 μl BSA (6%). Thereafter, the tube was centrifuged at 10 000 rpm/10 min/4 °C before 200 μl of the supernatant was counted by liquid scintillation of a Pacard Tri-Carb 1900 TR (PerkinElmer). The sum of 14CO2 and CA was considered as total substrate uptake of glucose, whereas the sum of 14CO2, ASM and CA was considered as total substrate uptake of oleic acid.
6
A2B Adenosine Receptor Ligand Binding Assay
7
Glucose and Oleic Acid Uptake in Skeletal Muscle Cells
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Skeletal muscle cells (7000 cells/well) were cultured in 96-well CellBIND® microplates. d -[14C(U)]Glucose (0.5 μCi/ml, 100 μM, or 200 μM) or [1-14C]oleic acid (0.5 μCi/ml, 100 μM) was given during 4-h trapping as described previously (Wensaas et al. 2007 (link)). In brief, a 96-well UniFilter® microplate (PerkinElmer, Shelton, CT, USA), activated for the capture of CO2 by the addition of 1 M NaOH, was mounted on top of the 96-well plate. After incubation, the cells were washed and harvested in 0.1 M NaOH. The 14CO2 trapped in the filter and cell-associated (CA) glucose was measured with a 2450 MicroBeta2 scintillation counter (PerkinElmer). The sum of 14CO2 and remaining CA radioactivity was considered an estimation of total cellular uptake of substrate: CO2 + CA. All data were related to total cell protein concentration measured in the cell lysates by the Bio-Rad protein assay.
8
Quantifying Skeletal Muscle Fatty Acid Oxidation
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Skeletal muscle cells (7,000 cells/well) were cultured on 96-well CellBIND® microplates. [1-14C]oleic acid (0.5 µCi/ml, 100 or 400 µM) were given during 4 h trapping as described62 (link) with or without 24 h prelabelling. In brief, a 96-well UniFilter®-96 GF/B micro plate was mounted on top of the CellBIND® plate and CO2 production was measured in DPBS medium with HEPES (10 mM) and L-carnitine (1 mM) adjusted to pH 7.2-7.3. CO2 production was assessed using a 2450 MicroBeta2 scintillation counter (PerkinElmer). Data were related to total cell protein concentration measured in the cell lysates by the Bio-Rad protein assay.
9
Glucose Uptake Assay in Skeletal Muscle
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Skeletal muscle cells were cultured and differentiated on 96-well CytoStar-T ® plates.
Measurement of [1-14 C]deoxy-D-glucose (2 µCi/ml, 7 µmol/l) accumulation by scintillation proximity assay (SPA) were performed in DMEM with 5.5 mmol/l glucose but without phenol red. Cells were incubated (5% CO 2 atmosphere at 37°C) for 8 h, with measurements at 0, 2, 4, 6, and 8 h on a PerkinElmer 2450 Microbeta 2 scintillation counter (Waltham, MA, USA). The radiolabelled deoxyglucose taken up and accumulated by adherent cells will be concentrated close to the scintillator embedded in the bottom of each well in the plate, and therefore provide a stronger signal than the radiolabel dissolved in the medium. Cells were harvested in 0.1 mmol/l NaOH and samples were stored at -20°C before analysis of protein content.
Measurement of [1-14 C]deoxy-D-glucose (2 µCi/ml, 7 µmol/l) accumulation by scintillation proximity assay (SPA) were performed in DMEM with 5.5 mmol/l glucose but without phenol red. Cells were incubated (5% CO 2 atmosphere at 37°C) for 8 h, with measurements at 0, 2, 4, 6, and 8 h on a PerkinElmer 2450 Microbeta 2 scintillation counter (Waltham, MA, USA). The radiolabelled deoxyglucose taken up and accumulated by adherent cells will be concentrated close to the scintillator embedded in the bottom of each well in the plate, and therefore provide a stronger signal than the radiolabel dissolved in the medium. Cells were harvested in 0.1 mmol/l NaOH and samples were stored at -20°C before analysis of protein content.
10
In Vitro Erythropoietin Bioassay
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An in vitro bioassay was performed using the erythropoietindependent subline UT-7/EPO derived from a human erythroleukemia. 27 Cells were maintained in Iscove's modified Dulbecco's medium containing 10% heat-inactivated fetal calf serum supplemented with L-glutamine (2 mM), penicillin (50 U/mL), streptomycin (0.05 mg/mL), and 0.2 IU/mL epoetin. Cells were subcultured every 2-3 days and split 1:5 when they had reached a cell density of 2-5 Â 10 5 cells/mL. Two-fold dilutions of the epoetin samples ranging from 0.1 IU/mL to 0.00078 IU/mL were incubated with UT-7/EPO cells at a density of 0.5 Â 10 4 cells/well. The plates were incubated at 37 C, 5% CO 2 for 48 h, and 3H-thymidine (thymidine[methyl-3H] 1 mCi [37 MBq]/mL, PerkinElmer, Beaconsfield, United Kingdom) 0.5 mCi/well, diluted in assay medium, added for the last 4 h of the incubation period. The cells were harvested onto glass fiber filter mats using a micro 96 harvester (Molecular Devices, Wokingham, United Kingdom) and the radioactivity incorporated into DNA estimated by scintillation counting using a 2450 MicroBeta2 scintillation counter (PerkinElmer, Waltham, MA). Bioactivity estimates of the different preparations were derived relative to the epoetin standard (Third WHO International Standard [third WHO IS] for erythropoietin, recombinant, for bioassay, 11/170 available from NIBSC, United Kingdom).
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