Embryos at 5 days post-fertilization (dpf) were incubated with 0, 300, 600, or 1000 μM 2-(N-(7-Nitrobenz-2oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG, abcam), a fluorescent glucose mimetic, for 6 h at 28.5 °C. Embryos were immobilized using 3-amino benzoic acid ethyl ester (Tricaine) and imaged on a Lionheart FX at 4× (BioTek Instruments). Total retinal fluorescence images were compiled and quantified using Fiji69 (link).
2 nbdg
Manufactured by Abcam
Sourced in United States
2-NBDG is a fluorescent glucose analog that can be used to monitor glucose uptake and metabolism in cells. It is a non-radioactive alternative to 2-deoxy-d-glucose and provides a straightforward way to measure glucose transport activity.
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Lab products found in correlation
2 nbdg, by Beckman Coulter (2 mentions)
Pyruvate assay kit, by Abcam (1 mentions)
Lactate assay kit, by Abcam (1 mentions)
2 nbdg, by BD (1 mentions)
Ab65330, by Abcam (1 mentions)
Ab204702, by Abcam (1 mentions)
Ab65342, by Abcam (1 mentions)
Dxflex, by Beckman Coulter (1 mentions)
Seahorse xf24 extracellular flux assay kits, by Agilent Technologies (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Mitotracker deep red fm, by Thermo Fisher Scientific (1 mentions)
Fortessa, by BD (1 mentions)
Rotenone, by Merck Group (1 mentions)
Antimycin, by Merck Group (1 mentions)
Succinate, by Merck Group (1 mentions)
2 nbdg, by Thermo Fisher Scientific (1 mentions)
Fluorescence based glucose assay kit, by Abcam (1 mentions)
2 deoxy d glucose, by Merck Group (1 mentions)
L lactate assay kit 1, by Eton Bioscience (1 mentions)
Sodium l lactate, by Merck Group (1 mentions)
18 protocols using 2 nbdg
1
Measuring Glucose Uptake in Zebrafish Embryos
2
Quantification of Intracellular Glucose Uptake
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M. smegmatis cultured in 0.02% glucose-supplemented 7H9T was normalized to an OD600 of 1.0 in fresh medium and treated with a 5 µM concentration of the fluorescent glucose analogue 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG; Abcam, Cambridge, MA) for 2 h at 37°C with shaking. The cultures were centrifuged at room temperature for 5 min and 4,000 rpm and then washed twice with PBST. After normalizing to the wet weight, the pellets were extracted with chloroform-methanol (2:1) overnight. The organic extracts were separated from the cell suspension by centrifugation at room temperature for 15 min and 12,000 rpm and then treated with 1 volume of H2O for 15 min at room temperature. The aqueous and organic layers were separated from each other suspension by centrifugation at room temperature for 5 min at 12,000 rpm and then subjected to TLC using chloroform-methanol-H2O (80:20:2) and 1-propanol–ethyl acetate–water (6:1:3), respectively. The TLC fluorescence was recorded by the ImageQuant system (GE Healthcare) or developed using 5% H2SO4 in ethanol.
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3
Quantifying Glucose Uptake and Metabolites
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The fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3- diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) was applied to measure glucose uptake (Abcam, MA, USA). Cells were washed twice with PBS and incubated in glucose-free culture medium for 1 h at 37 °C. Subsequently, 100 μM 2-NBDG was added and incubated in the dark for 1 h. After incubation, the cells were collected and suspended in cell-based assay buffer. The levels of 2-NBDG uptake were determined by flow cytometry (Beckman, DxFLEX, CA, USA).
The levels of glucose-6-phosphate, lactate and pyruvate were measured using a high-sensitivity glucose-6-phosphate assay kit, a pyruvate assay kit and a lactate assay kit (all from Abcam, MA, USA), respectively, according to the manufacturer’s instructions. The absorbance was determined using a microplate reader at 450 nm for glucose-6-phosphate and at 570 nm for lactate and pyruvate.
The levels of glucose-6-phosphate, lactate and pyruvate were measured using a high-sensitivity glucose-6-phosphate assay kit, a pyruvate assay kit and a lactate assay kit (all from Abcam, MA, USA), respectively, according to the manufacturer’s instructions. The absorbance was determined using a microplate reader at 450 nm for glucose-6-phosphate and at 570 nm for lactate and pyruvate.
4
Glucose Uptake in Activated CD4+ Cells
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CD4+ cells were cultured in RPMI medium in anti-CD3 coated plates (0.5 mg/mL) and activated with IFNγ (50 ng/mL) and YM155 (0 and 10 nM) for 24 h. Cells were washed and starved in glucose-free RPMI-medium for 2 h and then supplemented with 2-NBDG (100 μM, Abcam). 2NBDG uptake was registered after 30 min using flow cytometry (Verse, BD) and quantified as the ratio of mean fluorescence intensity to baseline.
5
Glucose Uptake and Metabolite Quantification
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Glucose uptake was analyzed using the fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3- diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) (Abcam, ab204702, MA, USA). Cells in BioFlex® six-well plates were washed with PBS and incubated in glucose-free culture medium for 1 h at 37 °C. Subsequently, 2-NBDG was added to reach a final concentration of 100 μM, and the cells were further incubated for 1 h under mechanical stretching. After the treatment, cells were collected and suspended in a cell-based assay buffer. The levels of 2-NBDG uptake were determined using flow cytometry (Beckman, DxFLEX, CA, USA) at 488 nm.
The levels of glucose-6-phosphate, lactate, and pyruvate were measured using high-sensitivity assay kits (for glucose-6-phosphate, Sigma, MAK021, Taufkirchen, Germany; for pyruvate, Abcam, ab65342, MA, USA; for lactate, Abcam, ab65330, MA, USA) according to the manufacturer’s instructions, respectively. The absorbance was recorded using a microplate reader at 450 nm for glucose-6-phosphate and at 570 nm for lactate and pyruvate.
The levels of glucose-6-phosphate, lactate, and pyruvate were measured using high-sensitivity assay kits (for glucose-6-phosphate, Sigma, MAK021, Taufkirchen, Germany; for pyruvate, Abcam, ab65342, MA, USA; for lactate, Abcam, ab65330, MA, USA) according to the manufacturer’s instructions, respectively. The absorbance was recorded using a microplate reader at 450 nm for glucose-6-phosphate and at 570 nm for lactate and pyruvate.
6
Extracellular Acidification Rate Analysis
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In the study, we used Seahorse XF24 Extracellular Flux Assay Kits (Agilent Technologies) for determination of the extracellular acidification rate (ECAR). According to the manufacturer's protocols, ECAR was examined with a Seahorse XF glycolysis stress test kit. BCPAP and TPC-1 cells were seeded overnight into a Seahorse XF24 cell culture plate at a density 1 × 104 cells/well. After baseline measurements, Seahorse injection ports were loaded with glucose, oligomycin, and 2-DG at the indicated time points. ECAR data were evaluated by Seahorse XF-96 Wave software. To measure glucose uptake, PTC cells were incubated with 100 µg/ml 2-deoxy-2-[(7- nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG, Abcam, #ab235976) in glucose-free medium for one hour and the fluorescence was measured at excitation and emission wave lengths of 485nm and 535nm, respectively.
7
Mitochondrial Profiling of Microglia by Flow Cytometry
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For the mitochondrial activity experiments, the cell suspension was incubated with the mitochondrial probes MitoTracker Green FM (Invitrogen, Cat# M7514) and MitoTracker DeepRed FM (Invitrogen, Cat# 22426) for 30min at 37degrees before FACS acquisition. For the electron transport chain experiments (ETC), the cell suspension was incubated with the mitochondrial probe Tetramethylrhodamine TMRM (Abcam, Cat# ab228569) and fluorescent glucose analog 2-NBDG (Abcam, Cat# 235976) for 30min at 37degrees before FACS acquisition. For challenging the ETC, the cell suspension was incubated each 90 seconds by the following drugs: 0,5ul of 100uM Rotenone (Sigma), 2ul of 2.5M Succinate (Sigma) and 0.5ul of 1mM Antimycin (Sigma). Cytometry was performed on Fortessa (BD Bioscience) and analyzed with FlowJo v 10 (Treestar). Microglial cells were gated from CD45low-CD11b+ cells followed by singlet after forward and side scatter pattern.
8
Glucose Uptake Assay with 2-NBDG
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2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) (Cat. ab146200; Abcam) was dissolved in DMSO to yield a 20 mM stock solution. After a single-cell suspension was obtained from the harvesting protocol, cells were resuspended in 500 µl nonsupplemented Leibowitz media and incubated at 37°C with a final 2-NBDG concentration of 80 µM or DMSO for the vehicle control for 35 min at 37°C. Samples were washed 1.5 times with PBS + 1% serum before staining.
9
Fuc Modulates Adipocyte Glucose Uptake
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Glucose uptake by adipocytes was determined using a fluorescent derivative of glucose (2-NBDG; Abcam, Tokyo, Japan). Adipocytes were treated with Fuc for 24 h, followed by incubation in a glucose-free medium, containing 100 µM 2-NBDG and 100 nM insulin, for 30 min. After washing the cells with PBS, fluorescence was measured at a wavelength of 460/540 nm. Lipopolysaccharide (LPS), tumor necrosis factor-α(TNFα), and interferon-γ (IFNγ) (LTI) were used to induce insulin resistance in adipocytes [26 (link)]. Adipocytes pre-treated with Fuc for 4 h were treated with 20 mM Fuc, 1 µg/mL LPS, 10 ng/mL TNFα, and 10 ng/mL IFNγ for 20 h followed by 2-NBDG and insulin for 30 min.
10
2-NBDG Uptake in Liver Cells
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50–100 µM of 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG, Abcam) was reconstituted in RPMI media (without nutrient supplement). 100 µl of the reconstituted 2-NBDG was aliquoted onto 5 x 105 liver non-parenchyma cells in a 96 well flat bottom plate and cells were cultured for 45–60 min (5% CO2, 37°C). Cells were washed and stained for antigen specific CD8+ CXCR5+ T cells for flow cytometry. Data were collected and analyzed by flow cytometry as previously described.
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