Metabolic assays were performed in NB4 and NB4-MR4 cells using a Seahorse Bioscience XFe96 analyzer (Agilent Technology, Santa Clara, USA). Cells were seeded and incubated at a density of 6×104 cells/well using Cell-Tak coated 96-well tissue culture plates (Corning). Cells were incubated in unbuffered DMEM at 37 °C without CO2 for 1 h. To evaluate mitochondrial function, the oxygen consumption rate (OCR) was evaluated using the Seahorse Bioscience XF Cell Mito Stress Test (Agilent Technology). Mitochondrial oxidative phosphorylation (OXPHOS) was analyzed under basal conditions, in the presence of 2 µM oligomycin (Merck KGaA), 1 µM carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) (Merck KGaA), 0.5 mM rotenone/antimycin A (R/A) (Merck KGaA). The extracellular acidification rate (ECAR), which is indicative of glycolysis, was analyzed using the glycolysis rate assay under basal conditions and after the injection of 0.5 mM R/A and 50 mM 2-deoxyglucose (Merck KGaA). The ATP production was measured using the Agilent Seahorse XF Real-Time ATP Rate Assays (Agilent Technologies) according to manufacturer’s protocol. The injection of 2 µM oligomycin and 0.5 mM R/A enables the calculation of mitochondrial and glycolytic ATP production. All the experiments were performed in duplicate.
2 deoxyglucose
Manufactured by Merck Group
Sourced in United States, Germany, Sao Tome and Principe, France, United Kingdom, Canada, China, Israel
2-deoxyglucose is a synthetic glucose analog. It is a chemical compound that can be used in research and experimental settings. The primary function of 2-deoxyglucose is to inhibit glycolysis, the metabolic pathway that converts glucose into energy. This compound can be utilized as a tool to study cellular metabolism and energy production processes.
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Lab products found in correlation
Oligomycin, by Merck Group (47 mentions)
Rotenone, by Merck Group (28 mentions)
Antimycin a, by Merck Group (25 mentions)
Metformin, by Merck Group (9 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions)
Oligomycin a, by Merck Group (7 mentions)
L glutamine, by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Tunicamycin, by Merck Group (6 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (6 mentions)
D glucose, by Merck Group (6 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Dexamethasone (dex), by Merck Group (5 mentions)
Mg132, by Merck Group (5 mentions)
Cycloheximide (chx), by Merck Group (5 mentions)
Oligomycin, by Agilent Technologies (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Xf24 extracellular flux analyzer, by Agilent Technologies (4 mentions)
Thapsigargin, by Merck Group (4 mentions)
Sodium azide, by Merck Group (4 mentions)
180 protocols using 2 deoxyglucose
1
Metabolic Profiling of Leukemia Cells
2
Cell Culture Media and Pharmacological Reagents
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Cell culture media: DMEM (#11,965,092), RPMI1640 (#21,870,084) and L-Glutamine (#25,030,081) were purchased from ThermoFisher Scientific (Life Technologies). Oligomycin A, 2-deoxyglucose, Digoxin, Matrigel®, methylcellulose, DMSO, analytical grade acetonitrile and methanol were purchased from Merck Life Science UK (Sigma Aldrich). IACS-10759 [14 (link), 15 (link)] was purchased from Chemietek (Indianapolis, IN, US).
3
Metabolic Profiling of Mesothelioma Cells
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A Seahorse Bioscience XF24 Extracellular Flux Analyzer was used to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Logarithmically growing mesothelioma cell lines were maintained in normal complete growth media and seeded onto a gelatin-coated 24-well XF Flux Analyzer assay plate at 80,000 cells/well (NCI-H2373) or 40,000 cells/well (HP1 and MSTO.211H) 24 h prior to assay. These seeding numbers were determined based on the doubling time of the mentioned cell lines. Cells were switched to serum-free XF assay media (Seahorse Biosciences, Billerica, MA, USA) with 25 mM glucose, 1 mM sodium pyruvate, and 2 mM glutamine and placed in a CO2-free incubator at least 2 h before the assay. Multiple measurements were obtained at baseline and following injection, sequentially of glucose (10 mM), oligomycin (2 μM ), and 2-deoxyglucose (100 mM) (Merck Life Science, Milan, Italy) for ECAR measurement and oligomycin (1 μM), FCCP (0.25 μM), and rotenone (1 μM) (Merck Life Science, Milan, Italy) for OCR measurement. Values are reported as mpH/min for ECAR and pmoles/min for OCR.
4
PFKL-mEGFP as Glucosome Marker
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The plasmid expressing human PFKL with a monomeric form of enhanced green fluorescent protein (mEGFP) was prepared previously (PFKL-mEGFP)4 (link). We have employed PFKL-mEGFP as a marker of the glucosome. 2-Deoxyglucose was purchased from Sigma.
5
Isolation of Primary Mouse Cardiomyocytes
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Primary mouse ventricular cardiomyocytes were prepared from hearts of 1- to 2-day-old C57BL6 mice (Jackson Laboratories, Bar Harbor, ME, USA) as previously described [11 (link)]. Experiments were performed on the culture day 5–7. 2-deoxy-glucose, trypsin inhibitor, bovine serum albumin, laminin, penicillin, and vitamin B12 were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
6
Neutrophil Metabolic Reprogramming Assay
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Neutrophils were cultured at 5 million cells/mL for 2-20 h at 37°C, in normoxia (21% O2, 5% CO2) or hypoxia (1% (3kPa) O2, 5% CO2) in two types of pre-equilibrated RPMI media 1640 with 10% fetal bovine serum (FBS) (GIBCO) and 1% Penicillin/Streptomycin, differing in the presence or absence of glucose. In the absence of glucose, dialyzed glucose-free FBS was used. To study the effects of stimulation and metabolic inhibitors, neutrophils were cultured with (100ng/mL) LPS E.coli serotype R515 (Enzo), CP-91149 (Sigma-Aldrich), 2-deoxyglucose (Sigma-Aldrich), Oligomycin A (Sigma-Aldrich) and Etomoxir (10uM) (CNIO Carlos III Therapies) (Raud et al., 2018 (link); Yao et al., 2018 (link)), Fructose-1,6-bisphosphatase-1 Inhibitor (FBP1i) (Cambridge Bioscience), MB05032 (Tocris Bioscience), 3-Mercaptopicolinic acid (HCl) (3-MPA) (Cambridge Bioscience), Glutaminase Inhibitor II, BPTES (MERCK Chemical).
7
Treg Cell Metabolic Profile Analysis
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2.5 × 105 flow cytometry-sorted Treg cells were seeded on a 96-well Seahorse cell culture plate and analyzed on a Seahorse XF24 Analyzer (10 (link)). The following drugs and corresponding doses were loaded onto ports A, B, C, and D in the same order: Oligomycin (2.5 uM, Sigma-Aldrich cat no. 75351), CCCP (10 uM, Sigma-Aldrich cat no. C2759), Antimycin A/Piercidin (2 uM each, Sigma-Aldrich cat no. A8674 and 15379, respectively), and 2-deoxyglucose (25 mM, Sigma-Aldrich cat no. D8375).
8
Preparation and Storage of Experimental Compounds
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AICAR (Toronto Chemical Research) was prepared at a concentration of 1.3 × 10−1 M in distilled water and kept at − 20 °C until use. 2-deoxyglucose (Sigma) was prepared at a concentration of 10−1 M in distilled water and used immediately. SBI-0206965 (Sigma-Aldrich), A-769662 (Tocris Bioscience) and the different modulator candidates were prepared in DMSO (Sigma). NA (Sigma Aldrich) was prepared in a saline-ascorbic solution (0.9% NaCl/0.01% ascorbic acid), Ach and L-NAME (Sigma Aldrich) were prepared in 0.9% NaCl solution. A stock solution (10−2 M) was prepared for each of them and stored at − 20 °C until use (maximum 3 months).
9
Opa Proteoliposome Uptake in HeLa Cells
10
Mitochondrial Hydrogen Sulfide Donor Synthesis
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AP39, a novel mitochondria-targeted H2S donor, was designed and synthesized by Medicilon Inc., as described previously [19 (link), 20 (link)]. Antimycin A, 0.25% trypsin, 2-deoxyglucose, oligomycin, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), trichloroacetic acid, rotenone, 7-azido-4-methylcoumarin (AzMC), monobasic potassium phosphate, dibasic sodium succinate hexahydrate, adenosine 5′-triphosphate (ATP) disodium salt hydrate, fatty acid-free BSA, sodium hydrosulfide hydrate (NaSH·xH2O), N,N-diethyl-p-phenylenediamine sulfate, magnesium chloride, and iron(III) chloride (FeCl3) were purchased from Sigma-Aldrich Company. The monoclonal anti-Aβ antibody 6E10 was obtained from Covance Inc. (Princeton, NJ, USA). Drp1 was purchased from Novus Biologicals, Inc. Fis1 was purchased from Proteintech Group. Mfn1 and Mfn2 antibodies were purchased from Santa Cruz Biotech, and OPA-1 antibody was purchased from BD Transduction Laboratories. The secondary antibodies goat anti-mouse-HRP and donkey anti-rabbit-HRP antibodies were purchased from GE Healthcare. The Pierce BCA protein assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Dulbecco's modified Eagle's medium (DMEM), 1% glutamine, and 1% penicillin were obtained from Invitrogen. All other chemicals were purchased from Amresco (Solon, OH, USA).
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