Cell Meter™ 2-NBDG Glucose Uptake Assay Kit

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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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

Overview SDS Protocol

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 37^oC 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 37^oC 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).

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)	467
Emission (nm)	538

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 37^oC 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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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