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5 3h glucose

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[5-3H]glucose is a radiochemical compound that contains the tritium (3H) radioactive isotope. It is commonly used as a tracer in various biochemical and biological research applications to study glucose metabolism and related processes.

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Lab products found in correlation

2 deoxy d 1 14c glucose, by PerkinElmer (2 mentions) Glucose go assay kit, by Merck Group (1 mentions) Human insulin, by Merck Group (1 mentions) Ls6500 scintillation counter, by Beckman Coulter (1 mentions) 9 10 3h palmitate, by PerkinElmer (1 mentions) 5 3h glucose, by Hewlett-Packard (1 mentions) 1900 tr liquid scintillation analyzer, by Hewlett-Packard (1 mentions) Atplite, by PerkinElmer (1 mentions)

Close spelling variants of this product

D-[5-3H(N)]-glucose [5-3H]-D-glucose

16 protocols using 5 3h glucose

1

Measuring Glycolytic Rate via 3H-Glucose Conversion

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Glycolytic rate was measured by monitoring the conversion of 5-3H-glucose to 3H2O, as described [6 (link)]. Cells were washed with PBS once and resuspended in Krebs buffer. After a 30-min incubation, Krebs buffer containing glucose and [5-3H]glucose (PerkinElmer) was added to cells to make final concentration of 10 mM glucose containing [5-3H]glucose (10 μCi/ml) in 0.5 ml and incubated for 1 hrs at 37°. After 1 hrs, 0.1 ml of 0.2 M HCl was then added to the mixture to stop the reaction. Then 0.2 ml of the reaction mixture was transferred to a small open tube that is placed in a scintillation vial that already contained 0.5 ml water for 48 hrs. The amount of diffused 3H2O was determined by scintillation counting. Glucose consumption was determined using the Glucose (GO) Assay kit (Sigma) following the manufacturer’s instructions.
Wang J., Duan Z., Nugent Z., Zou J.X., Borowsky A.D., Zhang Y., Tepper C.G., Li J.J., Fienh O., Xu J., Kung H.J., Murphy L, & Chen H.W. (2016). Reprogramming metabolism by histone methyltransferase NSD2 drives endocrine resistance via coordinated activation of pentose phosphate pathway enzymes. Cancer letters, 378(2), 69-79.
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2

Glycolytic Flux Measurement in Cells

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Glycolytic flux of cells was measured by monitoring the conversion of 5-3H-glucose to 3H2O as previously described7 (link). Briefly, 2 × 105 cells/well (BOECs from HPAH or healthy controls, or control PAECs) were plated in a 48 well plate in normal medium (EGM-2 MV 10% FBS). 24 hours after seeding (or 48 to 72 hours post-transfection as indicated in figure legend), 5-3H-glucose (PerkinElmer Life Sciences Inc., Boston, MA, USA) was added to a final concentration of 0.5 μCi/well (0.0185 MBq/well). Samples were incubated for 2 hours at 37 °C in a humidified incubator under 5% CO2. Then, 200 μl/well of supernatant was collected and placed into glass vials containing hanging wells with filter paper soaked with H20. Vials were capped, sealed with rubber stoppers, and incubated for 2–3 days at 37 °C to reach equilibrium. During incubation, 3H2O generated by glycolysis diffused from the bottom of the glass vials to the filter paper carried by the hanging wells through evaporation, condensation and absorption. The filter paper was then transferred into scintillation vials containing 5 ml of scintillation liquid and counted in a scintillation counter. Appropriate 3H-glucose-only and H2O-only controls were included, enabling the calculation of H2O in each sample and thus the flux of glycolysis as described7 (link).
Caruso P., Dunmore B.J., Schlosser K., Schoors S., Santos C.D., Perez-Iratxeta C., Lavoie J.R., Zhang H., Long L., Flockton A.R., Frid M.G., Upton P.D., D’Alessandro A., Hadinnapola C., Kiskin F.N., Taha M., Hurst L.A., Ormiston M.L., Hata A., Stenmark K.R., Carmeliet P., Stewart D.J, & Morrell N.W. (2017). Identification of miR-124 as a major regulator of enhanced endothelial cell glycolysis in pulmonary arterial hypertension via PTBP1 and PKM2. Circulation, 136(25), 2451-2467.
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3

Quantifying Glycolysis Flux via 3H2O Production

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Glycolysis was measured as the production of 3H2O from 5-3H-glucose, which occurs during the step catalysed by enolase. Cells were incubated with 5.5 mM glucose containing 17 MBq/mmol 5-3H-glucose (Perkin Elmer) in DMEM without pyruvate, glutamine or glucose for 90 min in a total volume of 0.5mls. At 90 min, 0.2 mls of the medium was applied to a column (volume 1 ml) of Dowex-1-borate (prepared from Dowex-1-chloride by the method of Hammerstedt, 1973 (link)). Washing with 2 column volumes of water eluted the 3H2O which was counted for radioactivity (Tri-Carb 2800TR Liquid Scintillation Analyser).
Board M., Lopez C., van den Bos C., Callaghan R., Clarke K, & Carr C. (2017). Acetoacetate is a more efficient energy-yielding substrate for human mesenchymal stem cells than glucose and generates fewer reactive oxygen species. The International Journal of Biochemistry & Cell Biology, 88, 75-83.
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4

Cardiac Glycolytic Flux Measurement

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Hearts were excised and perfused in non-recirculating Langendorff mode with Krebs-Henseleit (KH) buffer (37°C; 5% CO2/95% O2; containing in mmol/L: 118.5 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2 and 5 mM glucose; pH 7.4). Glycolytic flux was assessed by measuring the amount of 3H2O released through the metabolism of exogenous [5-3H]glucose (Perkin Elmer), as described.14 (link), 15 (link) Glucose utilization was then normalized to total heart weight.
Gibb A.A., Epstein P.N., Uchida S., Zheng Y., McNally L.A., Obal D., Katragadda K., Trainor P., Conklin D.J., Brittian K.R., Tseng M.T., Wang J., Jones S.P., Bhatnagar A, & Hill B.G. (2017). Exercise-Induced Changes in Glucose Metabolism Promote Physiological Cardiac Growth. Circulation, 136(22), 2144-2157.
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5

Measurement of Cellular Metabolic Fluxes

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Glycolysis was measured as previously described9 (link). Briefly, subconfluent HDLECs cultured in 12-well plates were incubated with 1 ml/well EBM2 medium (containing appropriate amount of serum and supplement) with 80 μCi/mmol 5-3H-glucose (Perkin Elmer) for 2-3 hr.. Then 0.8 ml/well medium was transferred into glass vials with hanging wells and filter papers soaked with H2O. After incubation in a cell culture incubator for at least 2 days to reach saturation, filter papers were taken out and the amount of evaporated 3H2O was measured in a scintillation counter. Glucose oxidation, glutamine oxidation and fatty acid oxidation were measured essentially as reported9 (link). For measurement of glucose uptake, HDLECs were incubated with 2-[1-14C]- deoxy-D-glucose (2.5 μCi/ml, Perkin Elmer) for 10 min. before PBS washing (at least 3 times to get rid of all radioactive medium) and then lyzed with 500 μL 0.1 N NaOH. 400 μl NaOH cell lysate for each sample was transferred to scintillation vials containing scintillation liquid and measured.
Yu P., Wilhelm K., Dubrac A., Tung J.K., Alves T.C., Fang J.S., Xie Y., Zhu J., Chen Z., De Smet F., Zhang J., Jin S.W., Sun L., Sun H., Kibbey R.G., Hirschi K.K., Hay N., Carmeliet P., Chittenden T.W., Eichmann A., Potente M, & Simons M. (2017). FGF-dependent metabolic control of vascular development. Nature, 545(7653), 224-228.
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6

Measurement of Cellular Metabolic Fluxes

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Glycolysis was measured as previously described9 (link). Briefly, subconfluent HDLECs cultured in 12-well plates were incubated with 1 ml/well EBM2 medium (containing appropriate amount of serum and supplement) with 80 μCi/mmol 5-3H-glucose (Perkin Elmer) for 2-3 hr.. Then 0.8 ml/well medium was transferred into glass vials with hanging wells and filter papers soaked with H2O. After incubation in a cell culture incubator for at least 2 days to reach saturation, filter papers were taken out and the amount of evaporated 3H2O was measured in a scintillation counter. Glucose oxidation, glutamine oxidation and fatty acid oxidation were measured essentially as reported9 (link). For measurement of glucose uptake, HDLECs were incubated with 2-[1-14C]- deoxy-D-glucose (2.5 μCi/ml, Perkin Elmer) for 10 min. before PBS washing (at least 3 times to get rid of all radioactive medium) and then lyzed with 500 μL 0.1 N NaOH. 400 μl NaOH cell lysate for each sample was transferred to scintillation vials containing scintillation liquid and measured.
Yu P., Wilhelm K., Dubrac A., Tung J.K., Alves T.C., Fang J.S., Xie Y., Zhu J., Chen Z., De Smet F., Zhang J., Jin S.W., Sun L., Sun H., Kibbey R.G., Hirschi K.K., Hay N., Carmeliet P., Chittenden T.W., Eichmann A., Potente M, & Simons M. (2017). FGF-dependent metabolic control of vascular development. Nature, 545(7653), 224-228.
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7

Quantitative Glycolysis Measurement in Muscle Fibers

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Glycolysis was measured by monitoring the conversion of 5-3H-glucose to 3H2O, as described previously 20 (link). Briefly, TA muscle fibers were incubated in a medium that contained [5-3H]-glucose (PerkinElmer) at a specific activity of 10 μCi. Labeled glucose metabolized by glycolysis produces 3H2O that is released into the media. Following incubation for 3 h at 37°C, media was transferred to uncapped tubes containing 0.5 mL of 0.2 M HCl. The tube was transferred to a scintillation vial containing 1 mL of H2O such that the water in the vial and the contents of the tube were not allowed to mix. The vials were sealed, and diffusion was allowed for 24 h. The amount of diffused 3H2O was determined by scintillation counting.
Gherardi G., Nogara L., Ciciliot S., Fadini G.P., Blaauw B., Braghetta P., Bonaldo P., De Stefani D., Rizzuto R, & Mammucari C. (2018). Loss of mitochondrial calcium uniporter rewires skeletal muscle metabolism and substrate preference. Cell Death and Differentiation, 26(2), 362-381.
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8

Glycolytic rate measurement in T cell subsets

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Glycolytic rate was measured by quantifying the release of 3H2O from [5-3H]-glucose as previously described44 (link). In brief, differentiated Th1, Th17 and Treg cells were collected and washed with glucose-free Krebs buffer (115 mM NaCl, 2 mM KCl, 25 mM NaHCO3, 1 mM MgCl2, 0.25% BSA pH 7.4) and incubated in 24 well plate at a density of 2 × 106 cells/ml (500 µl/well) in the same buffer for 30 min at 37 °C with 5% CO2. After incubation, 1 µCi of [5-3H]-glucose (PerkinElmer, Boston, MA) mixed with 10 mM of glucose was added to each well and incubated for 1 h. The glycolysis reaction was then quenched with 500 µl of 200 mM HCl. 100 µl of the reaction was added to small PCR tubes and placed in scintillation vials containing distilled water, sealed with parafilm, and incubated for 4 days. After that, the PCR tubes were transferred to fresh scintillation vials (undiffused fraction) and scintillation cocktail was added to both the diffused fraction and the undiffused fraction. Radioactivity was measured by liquid scintillation counting.
Alwarawrah Y., Danzaki K., Nichols A.G., Fee B.E., Bock C., Kucera G., Hale L.P., Taylor G.A, & MacIver N.J. (2022). Irgm1 regulates metabolism and function in T cell subsets. Scientific Reports, 12, 850.
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9

Langendorff Heart Glycolytic Flux

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Hearts were excised and perfused in nonrecirculating Langendorff mode with Krebs-Henseleit buffer (37°C; 5% CO2/95% O2; containing in mmol/L: 118.5 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, and 5 glucose; pH 7.4). Glycolytic flux was assessed by measuring the amount of 3H2O released through the metabolism of exogenous [5-3H]glucose (Perkin Elmer), as described.14 (link),15 (link) Glucose utilization was then normalized to total heart weight.
Gibb A.A., Epstein P.N., Uchida S., Zheng Y., McNally L.A., Obal D., Katragadda K., Trainor P., Conklin D.J., Brittian K.R., Tseng M.T., Wang J., Jones S.P., Bhatnagar A, & Hill B.G. (2017). Exercise-Induced Changes in Glucose Metabolism Promote Physiological Cardiac Growth. Circulation, 136(22), 2144-2157.
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10

Measuring Cardiomyocyte Glycolysis Rates

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Glycolysis rates were measured as described in [37 (link)]. Briefly, adult cardiomyocytes cultured in 12-well plates were incubated with 1 mL/well freshly prepared culture medium containing 80 uCi/mmol [5-3H] glucose (Perkin Elmer) with or without 5 µg/mL insulin (Human, Sigma 19,278) for 2 ~ 3 h. Then, 0.8 mL/well was transferred into glass vials with hanging holders and filter papers soaked with water. After incubation at 37 °C and 5% CO2 for 3 days to reach saturation, filter papers were removed and placed into scintillation cocktail to detect the amount of evaporated 3H2O.
Li P., Newhardt M.F., Matsuzaki S., Eyster C., Pranay A., Peelor FF 3.r.d., Batushansky A., Kinter C., Subramani K., Subrahmanian S., Ahamed J., Yu P., Kinter M., Miller B.F, & Humphries K.M. (2022). The loss of cardiac SIRT3 decreases metabolic flexibility and proteostasis in an age-dependent manner. GeroScience, 45(2), 983-999.
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