After overnight fasting, all mice were anesthetized with isoflurane and placed on the temperature-controlled pad to maintain their rectal temperature between 36.8 °C and 37.5 °C. [1-13C]-glucose (Sigma-Aldrich, >99% pure) solution was prepared in saline at a concentration of 0.5 mol/L. The left jugular vein was dissected, and [1-13C]-glucose solution was constantly injected into the left jugular vein through the microinfusion pump for a 30-min period at 0.1 mL/kg/min rate, according to a previous study (Zhou et al. 2018 (link)). Then, the blood glucose contents in the tail vein before and after injection were determined using a handheld glucometer. After 15-min infusion, mice were sacrificed by decapitation, and the brain tissues were rapidly isolated and frozen in liquid nitrogen and then kept at −80 °C.
1 13c glucose
Manufactured by Merck Group
Sourced in United States, Germany
[1-13C]-glucose is a stable isotope-labeled form of glucose, where the carbon-13 isotope is specifically incorporated at the first carbon position. This product is commonly used as a tracer in metabolic studies and research applications that require the monitoring of glucose metabolism and related physiological processes.
Automatically generated - may contain errors
Lab products found in correlation
1 2 13c acetate, by Cambridge Isotopes (2 mentions)
1 13c glucose, by Bio-Rad (2 mentions)
1 2 13c acetate, by Bio-Rad (2 mentions)
Rodent tail vein catheter and restraining apparatus, by Braintree Scientific (2 mentions)
Hrp labeled secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Deuterium oxide, by Cambridge Isotopes (1 mentions)
Econo gradient pump, by Bio-Rad (1 mentions)
Q5 high fidelity 2x master mix, by New England Biolabs (1 mentions)
U 13c acetate, by Merck Group (1 mentions)
U 13c glucose, by Merck Group (1 mentions)
Inova spectrometer, by Agilent Technologies (1 mentions)
2 13c pyruvic acid, by Merck Group (1 mentions)
3 13c glutamine, by Merck Group (1 mentions)
Hypersense dnp polarizer, by Oxford Instruments (1 mentions)
Mnova, by Mestrelab Research (1 mentions)
Toluene, by Merck Group (1 mentions)
13c215n glycine, by Cambridge Isotopes (1 mentions)
Hexokinase, by Merck Group (1 mentions)
Phosphoglucose isomerase, by Merck Group (1 mentions)
Mstfa, by Merck Group (1 mentions)
12 protocols using 1 13c glucose
1
In Vivo Glucose Metabolism Tracking
2
Isotope-Labeled Glucose and Acetate Metabolism
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R-sodium lipoic acid was a gift from GeroNova Research. A constant infusion of [1-13C]-glucose and [1,2-13C]-acetate was performed by using the ECONO gradient pump (Bio-Rad Laboratories, Hercules, CA, USA) and Rodent tail vein catheter and restraining apparatus (Braintree Scientific). Deuterium oxide (99.9%) and [1,2-13C]-acetate (99%) (Cambridge Isotope Laboratories, Andover, MA, USA), [1-13C]-glucose (99%) (Sigma-Aldrich, St Louis, MO, USA) were used for the NMR experiments. Chemicals of the purest grade were used for all assays. The primary antibodies against β-actin (SC-1616), GLUT3 (SC-74399), GLUT4 (sc-1608), Na+/K+-ATPase (SC-58628), and HRP-labeled secondary antibodies were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA). Antibodies for Akt (9272) and p-Akt (Ser473) (9271) were purchased from Cell Signaling Technology (Danvers, MA, USA).
3
Isotope Labeling for Yarrowia lipolytica
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Yarrowia lipolytica ACA DC 50109 was used in the present. The strain was maintained at −80 °C on potato dextrose medium with 20% glycerol (w/v). The medium for seed culture and batch fermentations are similar to that reported previously [7 (link)]. For isotope labeled experiment, yeast extract was replaced by vitamins solution which contained (per liter): biotin 0.05 g; p-amino benzoic acid 0.2 g; nicotinic acid 1 g; Ca-pantothenate 1 g; 19 pyridoxine–HCl 1 g and thiamine–HCl 1 g. The medium was supplemented with 30 g/L glucose or glycerol and for mixed substrate cultivations 1:1 mixture of glucose and glycerol were added. For the labelled experiments, 100% 1-13C glucose, 99% atom (Sigma, Germany) or 20% U-13C6 glucose 99% atom (EURISO-TOP GmbH, Germany) or 20% U-13C3 glycerol (99% atom, Cortecnet, France) were used. For mixed substrate labelled experiments, 50% U-13C3 glycerol, 20% 1-13C glucose and 30% naturally labelled glucose were used.
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Yarrowia lipolytica ACA DC 50109 was used in the present. The strain was maintained at −80 °C on potato dextrose medium with 20% glycerol (w/v). The medium for seed culture and batch fermentations are similar to that reported previously [7 (link)]. For isotope labeled experiment, yeast extract was replaced by vitamins solution which contained (per liter): biotin 0.05 g; p-amino benzoic acid 0.2 g; nicotinic acid 1 g; Ca-pantothenate 1 g; 19 pyridoxine–HCl 1 g and thiamine–HCl 1 g. The medium was supplemented with 30 g/L glucose or glycerol and for mixed substrate cultivations 1:1 mixture of glucose and glycerol were added. For the labelled experiments, 100% 1-13C glucose, 99% atom (Sigma, Germany) or 20% U-13C6 glucose 99% atom (EURISO-TOP GmbH, Germany) or 20% U-13C3 glycerol (99% atom, Cortecnet, France) were used. For mixed substrate labelled experiments, 50% U-13C3 glycerol, 20% 1-13C glucose and 30% naturally labelled glucose were used.
4
Isotopically Labeled Metabolite Assay
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Glucose, [1-13C] Glucose, [U-13C] Glucose, [U-13C] acetate, and all other chemicals unless otherwise stated were purchased from Sigma Aldrich (St. Louis, MO). Q5 High Fidelity 2X Master Mix was obtained from New England Biolabs (Ipswich, MA), and all other enzymes were purchased through Thermo Scientific (Waltham, MA).
5
Multinuclear MRS of Cell Cultures
6
In vivo 13C Isotope Labeling
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[1-13C]glucose (99%) was purchased from Sigma-Aldrich (St Louis, MO, USA); [1,2-13C]acetate (99%) and D2O (99.9%) from Cambridge Isotope Laboratories (Andover, MA, USA); the rodent tail vein catheter and restraining apparatus from Braintree Scientific, Inc. (MO, USA); the constant infusion of [1-13C]glucose and [1,2-13C]acetate was carried out by using a pump from Bio-Rad Laboratories Inc. (CA, USA). All other chemicals were the purest grade available from Sigma-Aldrich.
7
Isotope-Labeled Sample Preparation for DNP
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[1-13C]Glucose (Sigma Aldrich), [13C3, 15N]alanine, [13C2, 15N]glycine and sodium [1-13C]pyruvate (Cambridge Isotopes) were ground by hand to obtain the RFP. The RRPs were prepared by dissolving 4-hydroxy-TEMPO-benzoate (Sigma Aldrich) in mixtures of toluene-d6 (Armar Chemicals), THF-d8 (Armar Chemicals), toluene and THF (Sigma Aldrich). The finely ground RFP were then impregnated with the RRP solution directly in the DNP sample holder with 3 μl of the RRP added per milligram of RFP. The impregnated mixture in the DNP sample holder was then sonicated for 10 s before insertion in the DNP polarizer that resulted in rapid freezing of the sample in liquid helium.
8
Synthesis of 13C-Labeled Rifamycin Precursors
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To prepare [1-13C]-F-6-P, the reaction mixture containing 5 mM [1-13C] glucose (Sigma, USA), 6.25 mM MgCl2, 12.5 mM ATP, 1 U hexokinase (Sigma, USA), 1 U phosphoglucose isomerase (Sigma, USA) was incubated at 28 °C for 16 h46 (link),47 (link). To prepare the 13C-labeled R-L, 5 mM [1-13C]-glucose, 8.75 mM MgCl2, 12.5 mM ATP, 1 U hexokinase, 1 U phosphoglucose isomerase, 10 μM Rif15a, 10 μM Rif15b, 200 μM R-S, and 0.5 mM ThDP were mixed and incubated at 28 °C for 16 h. To prepare the 13C-labeled rifamcyin B, [39-13C] R-L was first prepared as described above; after 8 h, 2 μM Rif16, 20 μM seFdx, 10 μM seFdR, and 1 mM NADPH were added into the reaction mixture, and the reaction continued for 16 h at 28 °C. Notably, the one-pot reaction was unsuccessful because NADPH would reduce R-S to R-SV, thus preventing the Rif15 catalyzed reaction.
9
Metabolic Labeling with 13C-Glucose Isotopes
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The labeling substrates, [1-13C]glucose (99 atom%), [1,2-13C]glucose (99 atom%), and [13C6]glucose (99 atom%), were purchased from Sigma-Aldrich. Derivatization agents TBDMS [N-methyl-N-(t-butyldimethylsilyl) trifluoroacetamide plus 1% t-butyl-dimethylchlorosilane] and MSTFA [N-methyl-N-(trimethylsilyl) trifluroacetamide] were also purchased from Sigma-Aldrich. Medium components and other reagents were purchased from Himedia and Sigma-Aldrich.
10
Isotopic Labeling of NSCs and Astrocytes
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For isotopic labelling, NSCs and astrocytes were cultured in 6-well plates with custom N2B27-medium without glucose and without glutamine, supplemented with 10 mM [1- 13 C]glucose (Sigma-Aldrich, 297046) and 2 mM glutamine for NSCs, or without glutamine supplementation for astrocytes.
Sampling was performed immediately after label administration and then at 3 h, 12 h, and 24 h (Figure 1a). Replicate samples of culture supernatants were collected, clarified (2009g for 10 min) and stored at -20 °C for later extracellular metabolite analysis (see below). Upon removing the culture supernatants, cell monolayers were washed twice with ice-cold PBS to eliminate tracer amounts of extracellular metabolites, and the plates were placed on liquid nitrogen to rapidly stop metabolism. For intracellular metabolite extraction, 700 ll of ethanol 70% (v/v) were added to each well and the cells were detached using a cell scraper, followed by clarification (15 min at 20,0009g) and storage of the supernatants at -80 °C until GC-MS analysis (see below). The pellets containing cellular material were stored at -20 °C for protein quantification (see below). Additional wells seeded with NSCs and astrocytes were used to profile cell concentration and viability along the same sampling schedule.
Sampling was performed immediately after label administration and then at 3 h, 12 h, and 24 h (Figure 1a). Replicate samples of culture supernatants were collected, clarified (2009g for 10 min) and stored at -20 °C for later extracellular metabolite analysis (see below). Upon removing the culture supernatants, cell monolayers were washed twice with ice-cold PBS to eliminate tracer amounts of extracellular metabolites, and the plates were placed on liquid nitrogen to rapidly stop metabolism. For intracellular metabolite extraction, 700 ll of ethanol 70% (v/v) were added to each well and the cells were detached using a cell scraper, followed by clarification (15 min at 20,0009g) and storage of the supernatants at -80 °C until GC-MS analysis (see below). The pellets containing cellular material were stored at -20 °C for protein quantification (see below). Additional wells seeded with NSCs and astrocytes were used to profile cell concentration and viability along the same sampling schedule.
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