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Cell Meter™ 2-NBDG Glucose Uptake Assay Kit

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 37<sup>o</sup>C 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.
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 37<sup>o</sup>C 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.
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.
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.
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Catalog Number23500
Unit Size
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Excitation (nm)467
Emission (nm)538
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


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

Excitation488 nm laser
Emission530/30 nm filter
Instrument specification(s)FITC channel

Fluorescence microscope

ExcitationFITC filter
EmissionFITC filter
Recommended plateBlack wall/clear bottom

Components


Component A: 2-NBDG 1 vial (100 μL)
Component B: Assay Buffer I1 bottle (50 mL)
Component C: Assay Buffer II1 bottle (20 mL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells with your test compounds
  2. Add 2-NBDG staining solution
  3. Incubate cells  at 37oC for 20 minutes
  4. Remove 2-NBDG staining solution
  5. Wash cells with Assay Buffer I
  6. 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

  1. 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.

  2. At the end of the treatment, centrifuge the plate for 5 minutes at 800 rpm with brake off prior to your experiment.

  3. Aspirate the supernatant without disturbing cells.

  4. 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.

  5. At the end of the incubation, centrifuge the plate for 5 minutes at 800 rpm.

  6. Remove 2-NBDG staining solution without disturbing cells.

  7. 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.

  8. 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).

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)467
Emission (nm)538

Citations


View all 2 citations: Citation Explorer
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
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

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
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
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
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
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
2-NBDG, a fluorescent analogue of glucose, as a marker for detecting cell electropermeabilization in vitro
Authors: Raeisi E, Mir LM.
Journal: J Membr Biol (2012): 633
Uptake of 2-NBDG as a method to monitor therapy response in breast cancer cell lines
Authors: Millon SR, Ostr and er JH, Brown JQ, Raheja A, Seewaldt VL, Ramanujam N.
Journal: Breast Cancer Res Treat (2011): 55
Syntheses of 2-NBDG analogues for monitoring stereoselective uptake of D-glucose
Authors: Yamamoto T, Tanaka S, Suga S, Watanabe S, Nagatomo K, Sasaki A, Nishiuchi Y, Teshima T, Yamada K.
Journal: Bioorg Med Chem Lett (2011): 4088
Uptake of 2-NBDG by human breast cancer cells in vitro
Authors: Hu H, Shan XH, Zhu W, Qian H, Xu WR, Wang YF.
Journal: Zhonghua Zhong Liu Za Zhi (2010): 507
2-NBDG as a fluorescent indicator for direct glucose uptake measurement
Authors: Zou C, Wang Y, Shen Z.
Journal: J Biochem Biophys Methods (2005): 207