Cell Meter™ 2-NBDG Glucose Uptake Assay Kit
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Additional ordering information
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Spectral properties
Excitation (nm) | 467 |
Emission (nm) | 538 |
Storage, safety and handling
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Related products
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See also: Screen Quest™ Products
Excitation (nm) 467 | Emission (nm) 538 |
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.
Platform
Flow cytometer
Excitation | 488 nm laser |
Emission | 530/30 nm filter |
Instrument specification(s) | FITC channel |
Fluorescence microscope
Excitation | FITC filter |
Emission | FITC filter |
Recommended plate | Black wall/clear bottom |
Components
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).
Images

Figure 1. Fluorescence images of 2-NBDG uptake in CHO-K1 cells using Cell Meter™ 2-NBDG Glucose Uptake Assay Kit. CHO-K1 cells at 40,000 cells/well/100 µL were seeded overnight in a 96-well black wall/clear bottom plate. Cells were treated with 20 mM Glucose (B) or 100 µM Phloretin (C) at 37oC for 1 hour, then incubated with 100 µM 2-NBDG staining solution for 20 minutes. Untreated control cells were stained under the same conditions. The fluorescence signal was measured using a fluorescence microscope with FITC filter.

Figure 2. The Assay principle of 2-NBDG uptake in cells. 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. The fluorescence intensity is proportional to the cell glucose uptaking activity.

Figure 3. Flow cytometry of 2-NBDG uptake in CHO-K1 cells using Cell Meter™ 2-NBDG Glucose Uptake Assay Kit. CHO-K1 cells were treated with or without 100 µM Phloretin at 37 ºC for 1 hour, then incubated with 100 µM 2-NBDG staining solution for 20 minutes. To prepare adherent CHO-K1 cells for flow cytometry, EDTA was used to detach cells after staining. Fluorescence intensity was measured using ACEA NovoCyte flow cytometer in FITC channel.
Citations
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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)
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
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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
Glutamine promotes ovarian cancer cell proliferation through the mTOR/S6 pathway
Authors: Yuan, Lingqin and Sheng, Xiugui and Willson, Adam K and Roque, Dario R and Stine, Jessica E and Guo, Hui and Jones, Hannah M and Zhou, Chunxiao and Bae-Jump, Victoria L
Journal: Endocrine-related cancer (2015): 577--591
Authors: Yuan, Lingqin and Sheng, Xiugui and Willson, Adam K and Roque, Dario R and Stine, Jessica E and Guo, Hui and Jones, Hannah M and Zhou, Chunxiao and Bae-Jump, Victoria L
Journal: Endocrine-related cancer (2015): 577--591
References
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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
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Journal: J Mol Neurosci (2015): 126
Authors: Chen Y, Zhang J, Zhang XY.
Journal: J Mol Neurosci (2015): 126
2-NBDG fluorescence imaging of hypermetabolic circulating tumor cells in mouse xenograft model of breast cancer
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Journal: J Fluoresc (2013): 213
Authors: Cai H, Peng F.
Journal: J Fluoresc (2013): 213
Syzygium aqueum leaf extract and its bioactive compounds enhances pre-adipocyte differentiation and 2-NBDG uptake in 3T3-L1 cells
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Application notes
A New Red Fluorescent & Robust Screen Quest™ Rhod-4™ Ca2+Indicator for Screening GPCR & Ca2+ Channel Targets
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets
A Non-Radioactive Photometric Assay for Glucose Uptake in Insulin-Responsive 3T3-L1 Adipocytes
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Abbreviation of Common Chemical Compounds Related to Peptides
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets
A Non-Radioactive Photometric Assay for Glucose Uptake in Insulin-Responsive 3T3-L1 Adipocytes
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
Abbreviation of Common Chemical Compounds Related to Peptides