Glucose and glutamine oxidation fluxes were determined by the rate of 14CO2 released from 14C-U-glucose and 14C-U-glutamine, respectively. The Jurkat cells were treated for 48 h with 0.5 μM MPA or 5 μM rapamycin. Then, 5 × 106 cells were resuspended in 950 μL of Krebs-Ringer phosphate buffer supplemented with either 5 mM 14C-U-glucose (11 GBq/mmol, isotopic dilution 1/1000, Perkin Elmer) or 4 mM 14C-U-glutamine (9.69 GBq/mmol, isotopic dilution 1/1000, Perkin Elmer). After a 90-min incubation at 37 °C, the reaction was stopped by adding 250 μL of 6 N H2SO4, and CO2 was recovered for 1 h in benzethonium hydroxide. The radioactive CO2 was quantified by using liquid scintillation (Ultima Gold, Perkin Elmer).
U 14c glutamine
Manufactured by PerkinElmer
[U-14C]-glutamine is a radiolabeled amino acid used in research applications. It contains a carbon-14 isotope that is uniformly labeled across the glutamine molecule. This product is intended for use in scientific research, with specific applications determined by the researcher.
Automatically generated - may contain errors
Lab products found in correlation
U 14c glucose, by PerkinElmer (3 mentions)
Uridine, by Merck Group (2 mentions)
Apo transferrin, by Merck Group (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Insulin, by Merck Group (2 mentions)
Puromycin, by Merck Group (2 mentions)
Hydrocortisone, by Merck Group (2 mentions)
Ultima gold, by PerkinElmer (1 mentions)
Hyamine hydroxide, by PerkinElmer (1 mentions)
Liquid scintillation counter, by PerkinElmer (1 mentions)
Ammonium formate, by Thermo Fisher Scientific (1 mentions)
Cerulenin, by Merck Group (1 mentions)
Fluvastatin sodium hydrate, by Merck Group (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Nd 646, by MedChemExpress (1 mentions)
Isopropanol, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
8 protocols using u 14c glutamine
1
Measuring Cellular Metabolism Fluxes
2
Measuring Glutamine Oxidation Flux
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Glutamine oxidation flux was measured as previously described (53 (link)). Briefly, subconfluent HDLECs cultured in 6-well plates were incubated with 2 ml per well EBM2 medium (containing appropriate amounts of serum and supplement) with [14C(U)]-glutamine (PerkinElmer) for 6 h. Then, the cells were lysed using 12% perchloric acid, and the wells were covered immediately using filter papers soaked with hyamine hydroxide (PerkinElmer). After incubation in a fume hood for at least 12 h to reach saturation, filter papers were taken out, and the amount of evaporated 14CO2 was measured in a scintillation counter.
3
Measuring Cellular Glutamine Oxidation
4
Glutamine Uptake Assay in Cell Lines
5
Profiling Metabolic Pathways in Cell Lines
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Na2S, nonahydrate (99.99% purity), glucose, hydrocortisone, insulin, apo-transferrin, sodium selenite, sodium orthovanadate, uridine (cell culture grade), Protease Inhibitor Cocktail for use with mammalian cell and tissue extracts, puromycin, cerulenin, fluvastatin sodium hydrate, doxycycline, RIPA buffer, HPLC grade tert-Butyl methyl ether (MTBE), and chloroform were from Sigma. TOFA and ND-646 were from MedChemExpress. Dulbecco's modified Eagle's medium (DMEM) (with 4.5 g/l glucose, glutamine, and 110 mg/l sodium pyruvate), RPMI 1640 with glutamine, medium 199, fetal bovine serum (FBS), penicillin/streptomycin mixture, trypsin, EDTA, PBS, and Dulbecco's phosphate-buffered saline medium (DPBS) were from Gibco. Geneticin was purchased from Life Technologies. Hepes and LC-MS grade acetonitrile, methanol, water, isopropanol, and ammonium formate were from Fisher, and Eagle's minimal essential medium (EMEM) was from Lonza. [U-14C]-glucose (263.0 mCi/mmol) and [U-14C]-glutamine (281.0 mCi/mmol) were from PerkinElmer. EquiSPLASH lipid standard (#330731) was purchased from Avanti Polar Lipids, Inc.
6
Metabolic Profiling of Cancer Cells
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Unless specified, all reagents were obtained from Sigma and all the antibodies were from Santa Cruz Biotechnology, except for anti-IL-6 (AbCam). Matrigel Matrix was purchase from BD Biosciences. [U-14C] lactate, [U-14C] glucose and [U-14C] glutamine were from Perkin Elmer. All kits used to perform miRNA extraction and quantitative reverse transcriptase PCR were bought from Qiagen. c-Myc siRNA (sc-29226) and Control siRNA-A (sc-37007) were from Santa Cruz. Lipofectamine 2000 and Lipofectamine RNAiMAX Reagent were from Invitrogen. Metformin was obtained from Sigma. Glutaminase Inhibitor (compound 968) was purchased by Calbiochem. Molecular Clip and Tweezer were provided by Prof. T. Schrader (University of Duisburg-Essen, Germany); DASA-58 was synthesized by Prof. C. Nativi and B. Richichi, Department of Biochemistry, University of Florence.
7
Mammalian Cell Culture Reagents
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Na2S nonahydrate (99.99% purity), glucose, hydrocortisone, insulin, apo-transferrin, sodium selenite, sodium orthovanadate, uridine (cell culture grade), protease inhibitor cocktail for mammalian tissue extract, puromycin (Sigma P8833), and RIPA buffer were from Sigma. Monobromobimane (FluoroPure grade) was from Molecular Probes. [1-14C]-glucose (56.5 mCi/mmol), [6-14C]-glucose (60.3 mCi/mmol), and [U-14C]-glutamine (281.0 mCi/mmol) were from PerkinElmer. Cell culture media (DMEM with 4.5 g/l glucose, glutamine, and 110 mg/l sodium pyruvate [Cat. # 11995], RPMI 1640 with glutamine [Cat. # 11875], 199 [Cat. # 11150]), fetal bovine serum (FBS) (Cat. # 10437), penicillin/streptomycin mixture (Cat. # 15140), PBS (Cat. # 10010), and DPBS (Cat. # 14040) were from Gibco.
8
Metabolic Flux Analysis of ECs
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ECs were incubated in growth medium supplemented with [U-14 C]acetate (Perkin Elmer) for 24 hr following snap freezing and methanol-water-chloroform extraction. Phase separation was achieved by centrifugation at 4 C and the methanol-water phase containing polar metabolites was used as negative control. Chloroform phase containing fatty acids was added to scintillation liquid and counts were normalized to protein concentrations determined of the dried protein interphase.
14 C-glucose Oxidation Cells were incubated for 6 hr in growth medium containing 100 mCi/mmol [6-14 C]D-glucose (Perkin Elmer). Thereafter, 250 mL of 2 M perchloric acid was added to each well to stop cellular metabolism and wells were immediately covered with a 1x hyamine hydroxidesaturated Whatman paper. Overnight absorption of 14 CO 2 released during oxidation of glucose into the paper was performed at room temperature, and radioactivity in the paper was determined by liquid scintillation counting. 14 C-glutamine Oxidation 14 C-glutamine Oxidation was performed similarly as glucose oxidation, except that 0.5 mCi/mL [U-14 C]glutamine (Perkin Elmer) as tracer was used.
14 C-glucose Oxidation Cells were incubated for 6 hr in growth medium containing 100 mCi/mmol [6-14 C]D-glucose (Perkin Elmer). Thereafter, 250 mL of 2 M perchloric acid was added to each well to stop cellular metabolism and wells were immediately covered with a 1x hyamine hydroxidesaturated Whatman paper. Overnight absorption of 14 CO 2 released during oxidation of glucose into the paper was performed at room temperature, and radioactivity in the paper was determined by liquid scintillation counting. 14 C-glutamine Oxidation 14 C-glutamine Oxidation was performed similarly as glucose oxidation, except that 0.5 mCi/mL [U-14 C]glutamine (Perkin Elmer) as tracer was used.
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