Cells were plated in 96-well Lumox plates (Sarstedt) in glucose-free DMEM media (ThermoFisher) with 150 µg/mL of 2-NBDG (Cayman Chemical) for 3 h. Media was removed and cells were washed in PBS and then lysed in 100µL of ice-cold lysis buffer containing 0.1 M potassium phosphate and 1% (v/v) Triton X-100 adjusted to pH 10 to release the fluorescent glucose analog (2-NBDG). The amount of 2-NBDG taken up by GBM cells in each well was quantified using a fluorometric plate reader (BioTek Synergy 2) using an excitation wavelength of 468 nm and an emission wavelength of 540 nm. A portion of the lysate (10 µL) was removed and analyzed for total protein content using the Micro BCATM assay kit and the fluorescent intensity was normalized to total protein concentration.
2 nbdg
Manufactured by Cayman Chemical
Sourced in United States
2-NBDG is a fluorescent glucose analog used for the detection and measurement of glucose uptake in cells. It functions as a tool for studying glucose metabolism in biological systems.
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Lab products found in correlation
Bodipy fl c16, by Thermo Fisher Scientific (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
Oligomycin, by Merck Group (4 mentions)
Cytoflex s with analysis software, by Beckman Coulter (4 mentions)
Accutase, by Innovative Cell Technologies (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
2 nbdg, by Thermo Fisher Scientific (4 mentions)
2 nbdg, by PerkinElmer (3 mentions)
Lactic acid assay kit, by Nanjing Jiancheng (3 mentions)
Mitotracker green, by Thermo Fisher Scientific (3 mentions)
2 nbdg, by Agilent Technologies (3 mentions)
Synergy 2, by Agilent Technologies (2 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (2 mentions)
Antimycin a, by Merck Group (2 mentions)
Rotenone, by Merck Group (2 mentions)
S0026, by Beyotime (2 mentions)
Fluostar omega microplate reader, by BMG Labtech (2 mentions)
Seahorse xfe96 analyzer, by Agilent Technologies (2 mentions)
Igd 11 26c 2a, by BioLegend (2 mentions)
Gr 1 rb6 8c5, by BD (2 mentions)
149 protocols using 2 nbdg
1
Quantification of Glucose Uptake in GBM Cells
2
Glucose Uptake Assay using 2-NBDG
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2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG, Cayman Chemical, Ann Arbor, MI, USA), a glucose analog fluorescently labeled at the two position, is a substrate for glucose transporters.36 (link) Cells were cultured in IIA medium with 10% FBS, starved with serum-free and phenol-free IIA medium overnight, pretreated with inhibitors for 15 min, and then stimulated with E2 (10 nM) for 24 h. Medium was changed to phenol-free serum-free glucose-free DMEM with 50 μmol/l 2-NBDG for 30 min. Internalization of 2-NBDG was monitored using confocal microscopy. 2-NBDG uptake was quantified using fluorescence intensity and normalized to control.
3
Glucose Uptake Assay in Hematopoietic Stem Cells
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For the in vitro 2-NBDG assay, sorted HSCs were exposed to 200 µM 2-NBDG (Cayman Chemical, Cat# 11046) for 30 min. HSCs were then centrifuged at 340 × g at 4 °C for 5 min, and the supernatant was removed. The uptake of 2-NBDG was measured using FACS Aria IIIu. As a negative control, HSCs were simultaneously exposed to 54 µg/ml phloretin or 20 µg/ml cytochalasin B with 2-NBDG.
For the in vivo 2-NBDG assay, C57BL/6 mice treated with PBS or 5-FU were subjected to an in vivo 2-NBDG assay as reported by Jun et al., 2021 (link). Mice received a bolus dose of 375 µg 2-NBDG intravenously and were euthanized by cervical dislocation after 1 hr. Mice were immediately placed on ice, and all subsequent cell preparation processes were performed while the cells were chilled on ice. The 2-NBDG positive cell fraction was detected by flow cytometry.
For the in vivo 2-NBDG assay, C57BL/6 mice treated with PBS or 5-FU were subjected to an in vivo 2-NBDG assay as reported by Jun et al., 2021 (link). Mice received a bolus dose of 375 µg 2-NBDG intravenously and were euthanized by cervical dislocation after 1 hr. Mice were immediately placed on ice, and all subsequent cell preparation processes were performed while the cells were chilled on ice. The 2-NBDG positive cell fraction was detected by flow cytometry.
4
Metabolic profiling of CD8+ T cells
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In extracellular flux assays, ECAR of CD8+ T cells was measured with a XF96 analyzer (Seahorse Bioscience). 4 × 105 freshly prepared CD8+ T cells from WT, Nfatc1f/f x CD4-cre and Nfatc2−/− mice were maintained overnight without stimulation or stimulated by αCD3/CD28. In parallel, the same number of CTL- cells were stimulated for a further day by αCD3/CD8. Upon counting, the cells were seeded in a plate pre-coated with poly-D-lysine (Sigma; 50 μg/ml) in XF Seahorse medium with glutamine alone. Then, 10 mM glucose was injected, followed by 1 μM oligomycin and 20 mM 2-deoxyglucose. Mito stress test assays for the determination of oxygen consumption rate (OCR) were performed in a similar way according the manufacturers protocol (Seahorse Bioscience). 2-NBDG (Cayman Chemical) assays were performed by incubation of cells with 50 μM 2-NBDG for 1 h at 37 °C followed by flow cytometry.
5
Glucose Uptake in Caco-2 Cells
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Glucose uptake by Caco-2 cells was determined by the previously described method (5) . Caco-2 cells were obtained from American Type Cell Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, NY, USA) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco) at 37°C with 5% CO2 in air. Caco-2 cells were cultured in a monolayer in a 96-well plate (Tissue culture test plate 96F: TPP, Switzerland). The cells were incubated for an additional 24 h in serum-free DMEM. Subsequently, the cells were washed with Na buffer [10 mM HEPES (pH 7.4), 140 mM NaCl, 20 mg/mL bovine serum albumin] and incubated in Na buffer for 15 min. After incubation, the cells were incubated in Na buffer with 50 µM 2-deoxy-2-[(7-nitro-2, 1, 3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG: Cayman Chemical, MI, USA) for 10 min and then washed twice with ice-cold phosphate buffered saline (PBS) to remove the 2-NBDG. Fluorescence of the cells containing 2-NBDG was detected by fluorescence microscopy (IX73: Olympus, Tokyo, Japan) and calculated by Image J ver. 1.43u (National Institutes of Health, USA).
6
2NBDG Uptake Assay in Adipocytes
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For 2NBDG uptake assay, adipocytes were differentiated in a 96-well plate, then glucose and serum starved for 40 min prior to incubation with 2NBDG (200 µM, Cayman Chemical, Cat# 11046) in PBS for 80 min. At endpoint, cells were washed with PBS and 2NBDG content was measured via plate reading at excitation 475 nm, emission 550.
7
Glucose Uptake Measurement Protocols
8
Cardiac Myocyte Glucose Uptake Assay
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Glucose uptake was measured in isolated fresh cardiac myocytes using a non-radioactive glucose uptake cell-based assay and a fluorescent D-analog, 1-N-7-(nitrobenz-2-oxa-1, 3-diazol-4-yl) amino-2-deoxy-D -glucose (2-NBDG), following manufacturer’s recommendations (Cayman Chemical, Ann Arbor, MI, United States). The cells were plated on a 96-well plate at a density of 5,000 cells/well in glucose-free Tyrode buffer and incubated with/without GGF2 or insulin (0.01 μM) for 30 min. Subsequently, 2-NBDG (100 μM) was added to each well. After a 10-min incubation with 2-NBDG, cells were washed with PBS (200 μL/well) twice, and fluorescence was measured using a microplate reader at excitation/emission wavelengths of 535/758 nM.
9
Genetic Modification and Cell Signaling
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The H2-Kb was amplified from C57BL/6 splenocytes and cloned into a pCDH-GFP vector by enzymes EcoRI and BamHI (NEB). The Acaca and Acly shRNAs were cloned into a pLKO.1-GFP vector. LPS, OVA257-264, OVA323-339, and OVA were purchased from InvivoGen. TOFA, C75, 2-DG, 2-NBDG, etomoxir, and BMS-303141 were purchased from Cayman Chemical. Rapamycin, KU0063794, PP242, and BPTES were from Selleck. Mitotracker and BODIPY were purchased from Invitrogen. UK5099, 25-HC, TMR-dextran, oligomycin, fluoro-carbonyl cyanide phenylhydrazone (FCCP), rotenone, and antimycin A were from Sigma. Antibodies for TSC1 (#4906), TSC2 (#4308), c-Myc (#5605), c-Fos (#2250), p-S6 (#4858), S6 (#2317), IRF1 (#8478), IRF5 (#4950), ACC1 (#3662), H3K9ac (#9649), p-ACLY (#4331), ACLY (#4332), p-IκBα (#2859), p-IKKα/β (#2697), p65 (#8242), and CREB (#9197) were from Cell Signaling Technology. Antibodies against H3K27ac (ab4729), H3K27me3 (ab6002), H3K9me3 (ab8898), and IKKα/β (ab178870) were purchased from Abcam. Antibody for IκBα (SC371) was from Santa Cruz Biotechnology. Antibody against GAPDH (AP0063) was from Bioworld.
10
Glucose Uptake, Lactate, and ATP Assays
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For glucose uptake assays, cells were incubated with 100 μM 2-NBDG (11046, Cayman) beginning 30 min before the end of the experimental time. The cells were then washed with phosphate-buffered saline (PBS), resuspended in ice-cold PBS, collected and analyzed cytofluorometrically by recording FL-1 fluorescence. For analysis of lactate concentration, the indicated cells were cultured for 48 h. Whole-cell lysis was then determined using a lactate assay kit (K627, BioVision) according to the manufacturer’s instructions. For measurement of ATP levels, whole-cell extracts from the indicated cells were lysed in the lysis buffer provided an ATP assay kit (S0026, Beyotime). Intracellular ATP was evaluated by luciferase activity according to the standard protocol described in the ATP assay kit.
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