Cells were cultured in DMEM/F12 medium (17.5 mM unlabeled glucose) supplemented with 7.5 mM [U13C6]-glucose (Cambridge Isotope Laboratories) for 48 hr and total ion chromatography of fatty acids was performed by stable isotope tracing using [U13C6]-glucose for 48 hr. Three independent replicates of 2 × 106 cells for each cell line were collected, and the cell pellets were suspended in 0.5 ml of water and lysed by sonication. Cell debris was separated by centrifugation and proteins precipitated by treating the clarified supernatant with 1 ml of cold acetone. The final supernatant was air-dried and the free glutamic acid was converted to its trifluoroacetamide butyl ester for GC-MS analysis (Lee, 1996 (link)). Rate of fatty acid synthesis is represented by Oleate C18:1/Palmitoleate C16:1 ratio, demonstrating that Nanog silencing reduces fatty acid chain elongation. In addition, CO2 production of TICs is very low, indicating that TLR4/NANOG induction in TICs inhibits oxygen consumption through inhibition of FA oxidation and TCA cycle entrance.
U 13c6 glucose
Manufactured by Cambridge Isotopes
Sourced in United States
[U-13C6]glucose is a stable isotope-labeled glucose compound, where all six carbon atoms are labeled with the 13C isotope. It is used as a tracer in metabolic studies and as a substrate for in vitro and in vivo experiments to investigate glucose metabolism pathways.
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Lab products found in correlation
U 13c5 glutamine, by Cambridge Isotopes (13 mentions)
U 13c3 lactate, by Cambridge Isotopes (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
5 13c glutamine, by Cambridge Isotopes (2 mentions)
Glutamine, by Merck Group (2 mentions)
Norvaline internal standard, by Cambridge Isotopes (2 mentions)
15nh4cl, by Cambridge Isotopes (2 mentions)
U 13c16 palmitate, by Cambridge Isotopes (2 mentions)
1 2 13c2 glucose, by Cambridge Isotopes (2 mentions)
Sodium bicarbonate, by Merck Group (2 mentions)
D5030, by Merck Group (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Lc ms grade acetonitrile, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Tib 71, by American Type Culture Collection (1 mentions)
Luna nh2, by Phenomenex (1 mentions)
67 protocols using u 13c6 glucose
1
Fatty Acid Synthesis and Metabolism in TICs
2
Stable Isotope Tracing of Fatty Acids
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MHCC97H cells were cultured in DMEM/F12 medium (17.5 mM unlabeled glucose) supplemented with 7.5 mM [U13C6]-glucose (Cambridge Isotope Laboratories) for 48 h, and total ion chromatography of fatty acids was performed by stable isotope tracing using [U13C6]-glucose for 48 h. Three independent replicates of 2 × 106 cells for each cell line were collected; the cell pellets were suspended in 0.5 ml of water and lysed by sonication. Cell debris was separated by centrifugation, and proteins were precipitated by treating the clarified supernatant with 1 ml of cold acetone. The final supernatant was air-dried and the free glutamic acid was converted to its trifluoroacetamide butyl ester for GC–MS analysis, as previous described [27 , 28 (link)].
3
Metabolic Profiling of Glucose Fate
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To assess the metabolic fate of glucose, iPS-ECs were cultured with 5 mM U-13C6-glucose (Cambridge Isotope Laboratories, CLM-1396-1) in DMEM without glucose and pyruvate (Thermo Fisher Scientific, 12307263) supplemented with 1% dialysed FBS, 1% penicillin-streptomycin and 1% l -glutamine for 7 h, as pilot studies indicated that cells reached isotopic steady-state by this time. For BVOs, a 3 h incubation with 5 mM U-13C6-glucose (Cambridge Isotope Laboratories) in DMEM without glucose and pyruvate (Thermo Fisher Scientific) supplemented with 1% penicillin-streptomycin and 1% l -glutamine was performed. The medium was then removed and 80:20 methanol: water (−80 °C, extraction solvent) was added to cells for 15 min at −80 °C for metabolic quenching. Cells were scraped in the extraction solvent and cell lysates were pipetted into Eppendorf tubes followed by a sonication step. Then, samples were centrifuged at 16,000 × g for 10 min at 4 °C to pellet debris.
The supernatant was transferred to a new tube and dried using a SpeedVac (Thermo Fisher Scientific, Savant, SPD131DDA). The dried pellet was resuspended in 50 µl chloroform /100 µl methanol/100 µl H2O. The top, polar fraction was further dried and stored at −80 °C until LC-MS analysis. All metabolite analyses were performed on three biological replicates.
The supernatant was transferred to a new tube and dried using a SpeedVac (Thermo Fisher Scientific, Savant, SPD131DDA). The dried pellet was resuspended in 50 µl chloroform /100 µl methanol/100 µl H2O. The top, polar fraction was further dried and stored at −80 °C until LC-MS analysis. All metabolite analyses were performed on three biological replicates.
4
Stable Isotope Labeling of RAW 264.7 Cells
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The mouse macrophage RAW 264.7 cell line was obtained from ATCC (ATCC© TIB-71™). Cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) Fetal Bovine Serum and 100 U/ml penicillin, 100 mg/ml streptomycin (Life Technologies, Darmstadt), and cultivated in an incubator at 37°C with 5% CO2. For stable isotope labeling experiments, cells were seeded 24 hours before the experiment in RPMI-1640 medium as above, with 12.5 mmol/L U-13C6-Glucose (Cambridge Isotope Laboratories, USA) substituted for unlabeled glucose.
5
Tracing Glutamine and Glucose Metabolism in DLBCL
6
Intracellular Metabolite Profiling using Labeled Glucose
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For metabolomic analysis, cells were incubated in medium containing [U-13C6] glucose (Cambridge Isotope Laboratories CML1396, Tewksbury, MA, USA) for 24 h. To extract intracellular metabolites, cells grown in 6-well plate were briefly rinsed with 2 ml of ice-cold 150 mM ammonium acetate (pH = 7.3), before addition of 1 ml of ice-cold 80% methanol. Cells were scraped and transferred into Eppendorf tubes and then 5 nM D/L-norvaline was added. After vortexing at maximum velocity, samples were spun at 20,000g for 5 min at 4 °C. Supernatant was then moved into a glass vial, dried using speedvac centrifuge, and reconstituted in 50 μl 70% acetonitrile. 5 μl of each sample was injected onto a Luna NH2 (150 mm × 2 mm, Phenomenex, Torrance, CA, USA) column. Samples were analyzed with an UltiMate 3000RSLC (Thermo Scientific, Waltham, MA, USA) coupled to a Q Exactive mass spectrometer (Thermo Scientific, Waltham, MA, USA). The Q Exactive was run with polarity switching (+3.00 kV/−2.25 kV) in full scan mode with an m/z range of 70–1050. Separation was achieved using 5 mM NH4AcO (pH 9.9) and ACN. The gradient started with 15% NH4AcO and reached 90% over 18 min, followed by an isocratic step for 9 min and reversal to the initial 15% NH4AcO for 7 min.
7
Metabolic Profiling of CAKI-1 Cells
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CAKI-1 cells were grown to approximately 80% confluence. Cells were supplemented with fresh 5.5 mM [U-13C6] glucose (Cambridge Isotope Laboratories). After 24-hour incubation, metabolites were extracted using cold 80% HPLC graded methanol. The sample was centrifuged at 20,000g for 10 minutes at 4°C, and the supernatant was dried under a vacuum. Pellets were reconstituted in solvent (water/methanol/acetonitrile, 2:1:1, v/v) and further analyzed by LC-MS as previously described (51 (link)).
8
Isotopomer Analysis of Tumor Glucose
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For isotopomer analysis of glucose metabolism, tumour-bearing mice were injected with a bolus dose of 20 mg [U-13C6]-glucose (Cambridge Isotope Laboratories) via tail vein. Mice were sacrificed 15 min after the last injection. Tumours were dissected rapidly, snap frozen and stored at −80 °C.
9
Optimized Mitochondrial Assays for Drug Screening
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[U13C6]-glucose was purchased from Cambridge Isotope Laboratories (Cat#: CLM-1396). Hydroxychloroquine sulfate was purchased from ACROS Organics. Ferrostatin-1 (Cat#: SML0583-5MG) was purchased from Sigma Aldrich. BisBenzimide H33342 trihydrochloride (Hoechst) (Cat#: 14533) was purchased from Sigma Aldrich. MitoTracker Red CMXRos (Cat#: M7512) and MitoTracker Green FM (Cat#: M7514) were purchased from Invitrogen. TMRM Assay Kit (Mitochondrial Membrane Potential) (Cat#: ab228569) was purchased from Abcam.
10
Stable Isotope Tracer Experiments for Metabolic Analysis
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For [U-13C16] palmitate tracer experiments, 2.5 mM [U-13C16] sodium palmitate (Cambridge Isotopes, USA) was dissolved in 150 mM sodium chloride solution at 70 °C, and 40 mL of palmitate solution was added into 50 mL of 0.34 mM ultra-fatty acid free BSA (Sigma‐Aldrich, USA) solution at 37 °C to conjugate [U-13C16] palmitate to BSA. 1 mM working BSA-conjugated [U-13C16] palmitate solution was prepared via adjusting the pH to 7.4 and diluting to 100 mL with 150 mM sodium chloride. Finally, 50 µM BSA-conjugated [U-13C16] palmitate and 1 mM carnitine (Sigma‐Aldrich, USA) were mixed with culture medium in 10% dialyzed FBS. For [U-13C6] glucose tracer experiments, tracer media consisted of glucose free DMEM medium (Gibco, USA) with 10% FBS, supplemented with [U-13C6] glucose (Cambridge Isotopes, USA). During tracer experiments, the medium was removed, cultured cells were rinsed with PBS, and tracer media added to the wells. Cells were cultured in tracer media for 24 - 48 h before metabolite extraction.
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