[U-13C6]-glucose, and unlabeled sucrose, glucose, glutamine, asparagine plant protease inhibitor cocktail, Ponceau-S, trypsin and all common buffer reagents were purchased from Sigma (Milwaukee, WI). SDS-Page reagents were purchased from Invitrogen, (Carlsbad, CA). MTBSTFA +1% TBDMCS were purchased from Thermo Scientific (Waltham, MA). Uniformly labeled 7% glucose was obtained as a custom order from Cambridge Isotope Laboratories.
U 13c6 glucose
Manufactured by Merck Group
Sourced in United States
[U-13C6]-glucose is a stable isotope-labeled glucose molecule where all six carbon atoms are substituted with the carbon-13 (13C) isotope. It is a valuable tool for researchers in the fields of metabolic studies, tracer experiments, and isotope labeling analyses.
Automatically generated - may contain errors
Lab products found in correlation
Glutamine, by Merck Group (3 mentions)
Glucose, by Merck Group (3 mentions)
Ponceau s, by Merck Group (2 mentions)
Trypsin, by Merck Group (2 mentions)
U 13c3 glycerol, by Merck Group (2 mentions)
Vanquish uhplc, by Thermo Fisher Scientific (2 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (2 mentions)
C18 column, by Phenomenex (2 mentions)
Mtbstfa 1 tbdmcs, by Thermo Fisher Scientific (1 mentions)
Avance 500 spectrometer, by Bruker (1 mentions)
Asparagine, by Merck Group (1 mentions)
Chymotrypsin, by Merck Group (1 mentions)
Thermolysin, by Merck Group (1 mentions)
Plant protease inhibitor cocktail, by Merck Group (1 mentions)
N methyl n tert butyldimethylsilyl trifluoroacetamide mtbstfa 1 tert butyldimethylchlorosilane tbdmcs, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Transit x2 dynamic delivery system, by Mirus Bio (1 mentions)
Alanine, by Merck Group (1 mentions)
18 protocols using u 13c6 glucose
1
Metabolic Flux Analysis Protocol
2
Isotope-Labeled Precursors for NMR Analysis
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Sodium [1,2-13C2]acetate and L-valine-d8 were purchased from Cambridge Isotope Laboratories, Inc. [U-13C6]Glucose, sodium [1-13C]propionate, and L-[methyl-13C]methionine were purchased from Sigma-Aldrich Co. LLC. 1H and 13C NMR spectra were obtained on a Bruker AVANCE 500 spectrometer in DMSO-d6 using the signal of the residual solvent signals (δH 2.50, δC 40.0) as an internal standard. The 2H NMR spectrum was obtained on a Bruker AVANCE 500 spectrometer in DMSO. Chemical shifts were referenced to the solvent signal (δH(D) 2.50). ESITOFMS were recorded on a Bruker microTOF focus.
3
Metabolite Analysis via Mass Spectrometry
4
Metabolic Profiling of Cellular Processes
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[U-13C6]glucose, sucrose,
glucose, glutamine, asparagine, plant protease inhibitor cocktail,
Ponceau-S, bovine serum albumin (BSA), trypsin, chymotrypsin, thermolysin,
proteinase K, and all common buffer reagents were obtained from Sigma
(St. Louis, MO). Sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS–PAGE) reagents were purchased from Life
Technologies Inc. (Carlsbad, CA), and derivatization reagent N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide
(MTBSTFA) + 1% tert-butyldimethylchlorosilane (TBDMCS)
was purchased from Thermo Scientific (Waltham, MA).
glucose, glutamine, asparagine, plant protease inhibitor cocktail,
Ponceau-S, bovine serum albumin (BSA), trypsin, chymotrypsin, thermolysin,
proteinase K, and all common buffer reagents were obtained from Sigma
(St. Louis, MO). Sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS–PAGE) reagents were purchased from Life
Technologies Inc. (Carlsbad, CA), and derivatization reagent N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide
(MTBSTFA) + 1% tert-butyldimethylchlorosilane (TBDMCS)
was purchased from Thermo Scientific (Waltham, MA).
5
Metabolic Analysis of Pklr Knockdown
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All experiments were conducted using an Agilent 7890 gas chromatograph coupled to a 5977 mass spectrometer and collected using Agilent MassHunter software. AML12 cells were seeded on 6-well plates and transfected with either scr or si RNA against Pklr using TransIT-X2 Dynamic Delivery System (Mirus Bio LLC, Madison, WI). The next day medium was removed; cells were washed and replaced with medium containing [U-13C6]-glucose (Sigma-Aldrich) and grown for 24 hours before being analyzed. Samples were derivatized and analyzed as described previously.61 (link)
6
LC-MS Metabolite Quantification Protocol
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LC-MS grade methanol (MeOH) formic acid (FA) and acetonitrile (ACN) were from Fisher Scientific (Pittsburgh, PA, USA). Water was of ultrapure grade (EMD Millipore Co., Billerica, MA, USA). Deuterated internal standards (IS) D5-Glutamic acid, D5-Phenylalanine and D4-Succinic acid were from Cambridge Isotope Laboratories (Tewksbury, MA, USA). [U-13C515N2]-Glutamine, [5-13C]-Glutamine and [1-13C]-Glutamine were from Cortecnet (Paris, France). Glutamine, glutamic acid, [U-13C6]-glucose, ornithine, fumaric acid, aspartic acid, alpha-ketoglutaric acid, alanine and lactic acid were from Sigma (Sigma-Aldrich, St. Louis, MO, USA). Commercial negative/positive calibration and reference (lock masses) solutions for the MS device were from Agilent Technologies (Santa Clara, CA, USA).
7
Metabolic Profiling of PDAC Cell Lines
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All chemical standards (glucose, sodium lactate, sodium pyruvate) and reagents were purchased from Sigma-Aldrich (Castle Hill, Australia), unless indicated otherwise. HPLC-grade methanol and chloroform were used. [U-13C6]-glucose was purchased from Sigma-Aldrich (Castle Hill, Australia). Succinic acid-d6 (99 atom % D) from MSD Isotopes (Montreal, Canada) was kindly provided by BMSF (UNSW Australia). Recombinant human TNF-α was purchased from R&D systems (Minneapolis, MN, USA). Dialysed foetal calf serum (Life Technologies) was kindly provided by Holst Lab (USYD Australia).
HEK 293 and the PDAC cell line PANC-1 cells were cultured in Dulbecco’s modified Eagle’s medium containing 1 and 4.5 g/L glucose (Sigma-Aldrich), respectively, supplemented with 10 % foetal bovine serum (FBS, Gibco) and penicillin-streptomycin (Gibco). HPDE cells (a kind gift from Dr. Darren Saunders, UNSW Australia) were cultured in keratinocyte serum-free media (Gibco) supplemented with epidermal growth factor (5 ng/ml) bovine pituitary extract (50 ug/ml) and penicillin-streptomycin (Gibco).
HEK 293 and the PDAC cell line PANC-1 cells were cultured in Dulbecco’s modified Eagle’s medium containing 1 and 4.5 g/L glucose (Sigma-Aldrich), respectively, supplemented with 10 % foetal bovine serum (FBS, Gibco) and penicillin-streptomycin (Gibco). HPDE cells (a kind gift from Dr. Darren Saunders, UNSW Australia) were cultured in keratinocyte serum-free media (Gibco) supplemented with epidermal growth factor (5 ng/ml) bovine pituitary extract (50 ug/ml) and penicillin-streptomycin (Gibco).
8
Metabolic Analysis of Hepatic Energy Homeostasis
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ATP and AMP content in liver tissues or hepatocyte cultures were measured using an ATP/ADP/AMP determination kit (A-125; State University of New York, Buffalo) following the manufacturer’s protocol. The amount was normalized to the protein content, which was determined using a protein assay (Bio-Rad Laboratories), and is presented as micromoles per gram protein. NAD+ and NADH levels in samples from liver and hepatocyte cultures were measured using a colorimetric NAD+/NADH assay kit (E2ND-100; BioAssay Systems). cAMP levels in primary hepatocytes were determined by using the Direct cAMP ELISA kit (Enzo Life Sciences) following the manufacturer’s procedures.
For mass isotopomer distribution analysis, primary hepatocytes cultured in glucose-free DMEM supplemented with dialyzed 10% FBS were uniformly labeled with 20 mM [U-13C]-6-glucose (Sigma-Aldrich) for 6 h. The samples were analyzed by gas chromatography/mass spectrometry to determine the contribution of glucose to tricarboxylic acid cycle metabolites. Data are presented as fraction of 13C-labeled species within a given sample. M + 0 corresponds to an unlabeled metabolite (for example, m + 2 represents an ion labeled with two 13Cs). Glucose flux analyses were performed at the Mouse Metabolic Phenotyping Center at Case Western University (Cleveland, Ohio), which is a facility sponsored by the National Institutes of Health.
For mass isotopomer distribution analysis, primary hepatocytes cultured in glucose-free DMEM supplemented with dialyzed 10% FBS were uniformly labeled with 20 mM [U-13C]-6-glucose (Sigma-Aldrich) for 6 h. The samples were analyzed by gas chromatography/mass spectrometry to determine the contribution of glucose to tricarboxylic acid cycle metabolites. Data are presented as fraction of 13C-labeled species within a given sample. M + 0 corresponds to an unlabeled metabolite (for example, m + 2 represents an ion labeled with two 13Cs). Glucose flux analyses were performed at the Mouse Metabolic Phenotyping Center at Case Western University (Cleveland, Ohio), which is a facility sponsored by the National Institutes of Health.
9
Evaluating Metabolite Leakage During Quenching
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To investigate the necessity of washing with NaCl, [U-13C6]glucose (Sigma-Aldrich, Saint Louis, USA) was added into the bacterial culture and mixed, and bacteria were quenched immediately and washed twice with 0.85% NaCl. The pellets were resuspended in the 0.85% NaCl solution, the remaining [U-13C6]glucose was concentrated using C18 cartridges, and the concentration was determined using UHPLC-TOF/MS. To evaluate the leakage of intracellular metabolites after quenching caused by NaCl washing, the NaCl washing solutions from different quenching approaches were collected and concentrated using C18 cartridges, and the metabolites were analyzed by UHPLC-TOF/MS. The positive control group sample was suspended in 0.85% NaCl and heated in a water bath at 80°C for 20 min. Unquenched bacteria were used as a negative control.
10
Isotopic Glucose Acquisition and Reagents
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[1-13C1]-Glucose and [6-13C1]-glucose were purchased from Cambridge Isotope Laboratories Inc. (Tewksbury, MA, USA), and [U-13C6]-glucose was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Other chemicals and enzymes used for in vitro assays were obtained from Sigma-Aldrich Co. and Merck KGaA (Darmstadt, Germany). H2O2 solutions were freshly prepared from a 30% (w/w) stock solution (Sigma-Aldrich Co.).
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