Cell Meter™ 2-NBDG Glucose Uptake Assay Kit
Glucose metabolism, a process which converts glucose into energy, is a primary source of energy supply in most organisms. 2-NBDG [2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose], a fluorescently tagged glucose tracer, has been proven to effectively monitor glucose transportation in cells, as 2-NBDG transports into cells by the same glucose transporters (GLUTs) as glucose. Once 2-NBDG is uptaken in cells, it undergoes phosphorylation at C-6 position to give 2-NBDG-6-phosphate, which is well retained within the cells. Compared to other glucose tracers, such as 2-DG or FDG, 2-NBDG allows in situ measurements of 2-NBDG with high temporal and spatial resolution at single cell level. AAT Bioquest's Cell Meter™ 2-NBDG Glucose Uptake Assay Kit provides a sensitive and non-radioactive assay for measuring glucose uptake in cultured cells. In this kit, Assay Buffer I is used to enhance the uptake and retention of 2-NBDG in cells, while Assay Buffer II can improve the signal-to-background ratio of 2-NBDG in the cells. The fluorescence signal can be monitored by fluorescence microscope or flow cytometer with a 488 nm laser and 530/30 nm emission filter (FITC channel). Cell Meter™ 2-NBDG Glucose Uptake Assay Kit is the most robust tool for monitoring glucose transporters.
Example protocol
AT A GLANCE
Protocol summary
- Prepare cells with your test compounds
- Add 2-NBDG staining solution
- Incubate cells at 37oC for 20 minutes
- Remove 2-NBDG staining solution
- Wash cells with Assay Buffer I
- Analyze cells using fluorescence microscope or flow cytometer with 530/30 nm filter (FITC channel)
Important notes
Thaw all the components at room temperature before starting the experiment.
PREPARATION OF WORKING SOLUTION
Add 5 µL of 2-NBDG (10 mg/mL) (Component A) to 1.5 mL of Assay Buffer I (Component B) and mix well to make 2-NBDG staining solution. Protect from light. Note: This 2-NBDG staining solution is stable for 1 hour at room temperature. As the optimal staining conditions may vary depending on different cell types, it’s recommended to determine the optimal concentration of Component A for each specific experiment.
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
- Add test compounds into the cells and incubate for a desired period of time (such as 24, 48 or 96 hours) in a 37°C, 5% CO2 incubator. For blank wells (medium without the cells), add the same amount of compound buffer. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density and incubation time. We incubated CHO-K1 cells with 20 mM Glucose for glucose competition assay, and 100 µM Phloretin for GLUTs inhibition assay. See Data Analysis for details.
- At the end of the treatment, centrifuge the plate for 5 minutes at 800 rpm with brake off prior to your experiment.
- Aspirate the supernatant without disturbing cells.
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of 2-NBDG staining solution. Note: Optimal incubation time will need to be determined for each cell line and for each specific experiment. We incubated CHO-K1 cells at 37oC with 100 µM 2-NBDG (~34 µg/mL) for 20 minutes to show sufficient glucose uptake. See Data Analysis for details.
- At the end of the incubation, centrifuge the plate for 5 minutes at 800 rpm.
- Remove 2-NBDG staining solution without disturbing cells.
- For fluorescence microscope: Wash cells with Assay Buffer I (Component B) once. Keep cells in 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Assay Buffer II (Component C). Monitor the fluorescence signal using a fluorescence microscope with FITC filter.
- For flow cytometer: Detach cells if required using EDTA and resuspend cells in 100 µL/sample of Assay Buffer I (Component B). Monitor the fluorescence signal using a flow cytometer with 530/30 nm filter (FITC channel).
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Citations
View all 6 citations: Citation Explorer
Enhancing $\beta$-Cell Function and Identity in type 2 diabetes: The Protective Role of Coptis deltoidea CY Cheng et Hsiao via Glucose Metabolism Modulation and AMPK Signaling Activation
Authors: Zhang, Shan and Zhang, Yueying and Wen, Zhige and Chen, Yupeng and Bu, Tianjie and Yan, Yanan and Ni, Qing
Journal: Phytomedicine (2024): 155396
Authors: Zhang, Shan and Zhang, Yueying and Wen, Zhige and Chen, Yupeng and Bu, Tianjie and Yan, Yanan and Ni, Qing
Journal: Phytomedicine (2024): 155396
SDHB reduction promotes oral lichen planus by impairing mitochondrial respiratory function
Authors: Zhang, Hui and Xu, Beiyun and Liu, Jin and Guo, Bin and Sun, Hongying and Yang, Qiaozhen
Journal: (2022)
Authors: Zhang, Hui and Xu, Beiyun and Liu, Jin and Guo, Bin and Sun, Hongying and Yang, Qiaozhen
Journal: (2022)
Young and undamaged recombinant albumin alleviates T2DM by improving hepatic glycolysis through EGFR and protecting islet $\beta$ cells in mice
Authors: Liu, Hongyi and Ju, Anji and Dong, Xuan and Luo, Zongrui and Tang, Jiaze and Ma, Boyuan and Fu, Yan and Luo, Yongzhang
Journal: (2022)
Authors: Liu, Hongyi and Ju, Anji and Dong, Xuan and Luo, Zongrui and Tang, Jiaze and Ma, Boyuan and Fu, Yan and Luo, Yongzhang
Journal: (2022)
IKCa channels control breast cancer metabolism including AMPK-driven autophagy
Authors: Gross, Dominic and Bischof, Helmut and Maier, Selina and Sporbeck, Katharina and Birkenfeld, Andreas L and Malli, Roland and Ruth, Peter and Proikas-Cezanne, Tassula and Lukowski, Robert
Journal: Cell death \& disease (2022): 1--14
Authors: Gross, Dominic and Bischof, Helmut and Maier, Selina and Sporbeck, Katharina and Birkenfeld, Andreas L and Malli, Roland and Ruth, Peter and Proikas-Cezanne, Tassula and Lukowski, Robert
Journal: Cell death \& disease (2022): 1--14
NT1014, a novel biguanide, inhibits ovarian cancer growth in vitro and in vivo
Authors: Zhang, Lu and Han, Jianjun and Jackson, Am and a L , undefined and Clark, Leslie N and Kilgore, Joshua and Guo, Hui and Livingston, Nick and Batchelor, Kenneth and Yin, Yajie and Gilliam, Timothy P and others, undefined
Journal: Journal of Hematology & Oncology (2016): 91
Authors: Zhang, Lu and Han, Jianjun and Jackson, Am and a L , undefined and Clark, Leslie N and Kilgore, Joshua and Guo, Hui and Livingston, Nick and Batchelor, Kenneth and Yin, Yajie and Gilliam, Timothy P and others, undefined
Journal: Journal of Hematology & Oncology (2016): 91
References
View all 14 references: Citation Explorer
Transport of a Fluorescent Analogue of Glucose (2-NBDG) versus Radiolabeled Sugars by Rumen Bacteria and Escherichia coli
Authors: Tao J, Diaz RK, Teixeira CR, Hackmann TJ.
Journal: Biochemistry (2016): 2578
Authors: Tao J, Diaz RK, Teixeira CR, Hackmann TJ.
Journal: Biochemistry (2016): 2578
2-NBDG as a marker for detecting glucose uptake in reactive astrocytes exposed to oxygen-glucose deprivation in vitro
Authors: Chen Y, Zhang J, Zhang XY.
Journal: J Mol Neurosci (2015): 126
Authors: Chen Y, Zhang J, Zhang XY.
Journal: J Mol Neurosci (2015): 126
Syzygium aqueum leaf extract and its bioactive compounds enhances pre-adipocyte differentiation and 2-NBDG uptake in 3T3-L1 cells
Authors: Manaharan T, Ming CH, Palanisamy UD.
Journal: Food Chem (2013): 354
Authors: Manaharan T, Ming CH, Palanisamy UD.
Journal: Food Chem (2013): 354
2-NBDG fluorescence imaging of hypermetabolic circulating tumor cells in mouse xenograft model of breast cancer
Authors: Cai H, Peng F.
Journal: J Fluoresc (2013): 213
Authors: Cai H, Peng F.
Journal: J Fluoresc (2013): 213
In vivo imaging of epileptic activity using 2-NBDG, a fluorescent deoxyglucose analog
Authors: Tsytsarev V, Maslov KI, Yao J, Parameswar AR, Demchenko AV, Wang LV.
Journal: J Neurosci Methods (2012): 136
Authors: Tsytsarev V, Maslov KI, Yao J, Parameswar AR, Demchenko AV, Wang LV.
Journal: J Neurosci Methods (2012): 136
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