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2-NBDG

(Synonyms: 2-(N-7-硝基-2,1,3-苯并恶二唑-4-氨基)-2-脱氧-D-葡萄糖) 目录号 : GC10289

2-NBDG 是一种荧光 D-葡萄糖类似物,是一种用于监测葡萄糖进入活细胞的情况的荧光指示剂。

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实验参考方法

Procedure for 2-NBDG uptake assay for MEFs [1]

1.        Mouse embryonic fibroblasts (MEFs) are isolated from the embryos of C57BL/6 WT mouse (13.5 days).

2.        Culture the MEF cells until reaching 80-90% confluence in 10 cm Petri dishes with DMEM growth medium in a humidified cell culture incubator (37 °C, 5% CO2).
Note: Don’t use MEFs beyond passage 3. MEFs usually become senescent at about passage 4 to 5.

3.        Remove culture medium and wash cells one time with 10 ml 1x PBS.

4.  Trypsinize cells using 4 ml of 0.05% trypsin-EDTA for 3 min at 37 °C.

Note: Use room temperature or pre-warmed 1x PBS from Step A3 to Step A9. Using chilled 1x PBS after Step A9.

5.  Transfer cells to 15 ml polystyrene centrifuge tubes.

6.  Harvest cells at 200 x g for 5 min by centrifugation.

7.  Wash pelleted cells one time with 5 ml 1x PBS.

8.  Count cells using a hemocytometer chamber.

9.  Incubate 1 x 106 MEF cells in a 37 °C water bath for 2 h with 1 ml of PBS containing 100 μM 2-NBDG. Incubate the same number of MEFs in the water bath with 1 ml PBS without 2-NBDG as a negative control.

10.             Pellet the cells at 200 x g for 5 min by centrifugation. After washing the cells with chilled 1x PBS, the cells are pelleted at 200 x g for 5 min by centrifugation.

11.             Resuspend cells in 0.5 ml of ice-cold 1x PBS with 2% FBS.
Note: Always keep cells on ice after this step.

12.             Filter cells through a 35 µm nylon mesh (the cell-strainer cap of the 5 ml round-bottom polystyrene tubes) to obtain a uniform single-cell suspension in a 5 ml tube.

13.             Keep the samples on ice until analysis on a flow cytometer.

14.             Perform flow cytometric analysis. Acquire 10,000 single-cell events per reaction.

15.             Analyze fluorescence intensity

 

Procedure for 2-NBDG uptake assay for breast cancer cells [1]

Using the same procedure as MEFs’ uptake assay except incubating 1 x 106 MCF7 cells in a 37 °C water bath only for 30 min (instead of two hours for MEFs) with 1 ml of PBS containing 100 μM 2-NBDG.

 

10 mM stock of 2-NBDG: Dissolve 5 mg 2-NBDG in 1.46 ml PBS. Store at -20 °C in the dark

 

This protocol only provides a guideline, and should be modified according to your specific needs

 

[1]. Dong, S., Baranwal, S., Garcia, A., Serrano-Gomez, S. J., Eastlack, S., Iwakuma, T., Mercante, D., Mauvais-Jarvis, F. and Alahari, S. K. (2017). Nischarin inhibition alters energy metabolism by activating AMP-activated protein kinase. J Biol Chem 292(41): 16833-16846.

产品描述

2-NBDG is a fluorescence-labeled 2-deoxy-glucose analog useful as a tracer for evaluation of cellular glucose metabolism (Ex/Em: 475/550 nm).

excitation and emission of 2-NBDG

 

Glucose is a necessary source of energy for sustaining cell activities and homeostasis in tissues. Glucose metabolism is an important target in many diseases and changed with the pathological condition, therefore, evaluation of glucose metabolism can be a significant indication in disease progressions.

 

2-NBDG can be used in many kinds of cells in vitro, such as HepG2 human hepatocarcinoma cells, L6 rat skeletal muscle cells, MCF-7 breast cancer epithelial cells and astrocytes, it is also used in disease models, epilepsy rat, hyperglycemia, diabetes or mouse xenograft model of cancer.

 

2-NBDG enters cells through glucose transporters and is subsequently phosphorylated by hexokinase and trapped inside cells. Flow cytometric detection of fluorescence produced by cells can be performed to examine 2-NBDG uptake into living cells, and the intracellular concentration of transported 2-NBDG can be measured with a fluorescence microplate assay. It can be detected with a fluorescence imaging microscopy or CCD camera simply as well.

 

2-NBDG is a fluorescently labeled glucose tracer that is transported into cells by the same glucose transporter (GLUT) as glucose. Once 2-NBDG is taken up by cells, it is phosphorylated at the C-6 position to give 2-NBDG-6-phosphate, which is well retained in the cell. Compared to other glucose tracers such as 2-DG or FDG, 2-NBDG enables in situ measurement of 2-NBDG with high temporal and spatial resolution at the single-cell level. (suitable for fluorescence microscopy and flow cytometry detection)

Principle of 2-NBDG

 

Rationale for 2-NBDG glucose uptake assay in cells: Once 2 NBDG is taken up by cells, it is phosphorylated at the C-6 position to generate 2-NBDG-6-phosphate in 2 NBDG metabolism, which is well retained in the cell, the fluorescence intensity is proportional to the cellular glucose uptake activity.

 

References:

[1]. Zou C, Wang Y, Shen Z. 2-NBDG as a fluorescent indicator for direct glucose uptake measurement[J]. Journal of biochemical and biophysical methods, 2005, 64(3): 207-215.

[2]. O’Neil R G, Wu L, Mullani N. Uptake of a fluorescent deoxyglucose analog (2-NBDG) in tumor cells[J]. Molecular Imaging and Biology, 2005, 7(6): 388-392.

[3]. Tsytsarev V, Maslov K I, Yao J, et al. In vivo imaging of epileptic activity using 2-NBDG, a fluorescent deoxyglucose analog[J]. Journal of neuroscience methods, 2012, 203(1): 136-140.

[4]. Yan Chen, Junjian Zhang, Xiang-yang Zhang, 2-NBDG as a Marker for Detecting Glucose Uptake in Reactive Astrocytes Exposed to Oxygen-Glucose Deprivation In Vitro. J Mol Neurosci (2015) 55:126–130.

[5]. Tsytsarev V, Maslov K I, Yao J, et al. In vivo imaging of epileptic activity using 2-NBDG, a fluorescent deoxyglucose analog[J]. Journal of neuroscience methods, 2012, 203(1): 136-140.

Chemical Properties

Cas No. 186689-07-6 SDF
别名 2-(N-7-硝基-2,1,3-苯并恶二唑-4-氨基)-2-脱氧-D-葡萄糖
化学名 (3R,4R,5S,6R)-6-(hydroxymethyl)-3-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)tetrahydro-2H-pyran-2,4,5-triol
Canonical SMILES OC[C@](O1)([H])[C@](O)([H])[C@@](O)([H])[C@](NC2=CC=C(N(=O)=O)C3=NON=C23)([H])C1([H])O
分子式 C12H14N4O8 分子量 342.26
溶解度 ≥ 17.1mg/mL in Water with ultrasonic 储存条件 Store at -20°C
General tips For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.
Shipping Condition Evaluation sample solution : ship with blue ice
All other available size: ship with RT , or blue ice upon request

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Research Update

Quantification of 2-NBDG, a probe for glucose uptake, in GLUT1 overexpression in HEK293T cells by LC-MS/MS

Anal Biochem2021 Oct 15;631:114357.PMID: 34469746DOI: 10.1016/j.ab.2021.114357

The growth and proliferation of most cancer cells involve the excessive uptake of glucose mediated by glucose transporters. An effective strategy for cancer therapy has been to inhibit the GLUTs that are usually overexpressed in a variety of tumor cells. 2-NBDG is a GLUT1 substrate that can be used as a probe for GLUT1 inhibitors. An accurate and simple assay for 2-NBDG in a HEK293T cell model overexpressing GLUT1 was developed using liquid chromatography-tandem mass spectrometry. Chromatographic separation was achieved using a Xbridge® Amide column (3.5 μm, 2.1 mm × 150 mm, Waters) with acetonitrile-water containing 2 μM ammonium acetate (80:20, v/v) at a flow rate of 0.25 mL/min. Mass detection was conducted in the parallel reaction monitoring (PRM) mode. The calibration curve for 2-NBDG showed good linearity in the concentration range of 5-500 ng/mL with satisfactory precision, a relative standard deviation ranging from 2.92 to 9.59% and accuracy with a relative error ranging from -13.14 to 7.34%. This method was successfully applied to quantify the uptake of GLUT1-mediated 2-NBDG, and the results clearly indicated inhibition of GLUT1 by WZB117 and quercetin (two potent glucose transporter inhibitors) in the GLUT1-HEK293T cell model. This study provides a convenient and accurate method for high-throughput screening of selective and promising GLUT1 inhibitors.

Efficient and modified 2-NBDG assay to measure glucose uptake in cultured myotubes

J Pharmacol Toxicol MethodsMay-Jun 2021;109:107069.PMID: 33892108DOI: 10.1016/j.vascn.2021.107069

Under type-2 diabetes, insulin resistance develops in skeletal muscles as a key defect and to study the disorder, its manifestation, and possible solution, measurement of glucose uptake is a fundamental necessity. Of various approaches (i.e. scintillation counting, flow cytometry, fluorometry and spectrophotometry) fluorescent labelled glucose analogue, 2-NBDG solution is the most popular one. Although 2-NBDG based assay is the most widely used approach in various cells including skeletal muscle, even then all available protocols possess huge variability which impacts the overall data reproducibility. Moreover, starvation (use of glucose/serum free medium), one of the prerequisite condition for glucose uptake assay, itself induces stress specifically during longer pre-incubation periods and alters muscle cell metabolism and morphology, but the fact has not been duly considered. Therefore in the present article, using specific skeletal muscle cells i.e. C2C12 myotubes, we have re-established the conditions like pre-incubation time period, concentrations of insulin, glucose and serum/BSA while maintaining the cultured myotubes in morphologically healthy state. Our lab standardized protocols were observed to be effective in studying insulin resistance condition induced by diverse stresses (oxidative & inflammation) in myotubes. Comparative study conducted with already established protocols demonstrates that the present method is more efficient, effective and better improvised for studying glucose uptake in C2C12.

Uptake of fluorescent D- and L-glucose analogues, 2-NBDG and 2-NBDLG, into human osteosarcoma U2OS cells in a phloretin-inhibitable manner

Hum Cell2021 Mar;34(2):634-643.PMID: 33454890DOI: 10.1007/s13577-020-00483-y

Mammalian cells take in D-glucose as an essential fuel as well as a carbon source. In contrast, L-glucose, the mirror image isomer of D-glucose, has been considered merely as a non-transportable/non-metabolizable control for D-glucose. We have shown that 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), a D-glucose analogue combining a fluorophore NBD at the C-2 position, is useful as a tracer for monitoring D-glucose uptake through glucose transporters (GLUTs) into mammalian cells. To more precisely evaluate the stereoselectivity of 2-NBDG uptake, we developed an L-glucose analogue 2-NBDLG, the mirror-image isomer of 2-NBDG. Interestingly, 2-NBDLG was taken up into mouse insulinoma MIN6 cells showing nuclear heterogeneity, a cytological feature of malignancy, while remaining MIN6 cells only exhibited a trace amount of 2-NBDLG uptake. The 2-NBDLG uptake into MIN6 cells was abolished by phloretin, but persisted under blockade of major mammalian glucose transporters. Unfortunately, however, no such uptake could be detected in other tumor cell lines. Here we demonstrate that human osteosarcoma U2OS cells take in 2-NBDLG in a phloretin-inhibitable manner. The uptake of 2-NBDG, and not that of 2-NBDLG, into U2OS cells was significantly inhibited by cytochalasin B, a potent GLUT inhibitor. Phloretin, but neither phlorizin, an inhibitor of sodium-glucose cotransporter (SGLT), nor a large amount of D/L-glucose, blocked the 2-NBDLG uptake. These results suggest that a phloretin-inhibitable, non-GLUT/non-SGLT, possibly non-transporter-mediated yet unidentified mechanism participates in the uptake of the fluorescent L-glucose analogue in two very different tumor cells, the mouse insulinoma and the human osteosarcoma cells.

Cellular binding and uptake of fluorescent glucose analogs 2-NBDG and 6-NBDG occurs independent of membrane glucose transporters

Biochimie2021 Nov;190:1-11.PMID: 34224807DOI: 10.1016/j.biochi.2021.06.017

The classical methods for determining glucose uptake rates in living cells involve the use of isotopically labeled 2-deoxy-d-glucose or 3-O-methyl-d-glucose, which enter cells via well-characterized membrane transporters of the SLC2A and SLC5A families, respectively. These classical methods, however, are increasingly being displaced by high-throughput assays that utilize fluorescent analogs of glucose. Among the most commonly used of these analogs are 2-NBDG and 6-NBDG, which contain a bulky 7-nitro-2,1,3-benzoxadiazol-4-yl-amino moiety in place of a hydroxy group on d-glucose. This fluorescent group significantly alters both the size and shape of these molecules compared to glucose, calling into question whether they actually enter cells by the same transport mechanisms. In this study, we took advantage of the well-defined glucose uptake mechanism of L929 murine fibroblasts, which rely exclusively on the Glut1/Slc2a1 membrane transporter. We demonstrate that neither pharmacologic inhibition of Glut1 nor genetic manipulation of its expression has a significant impact on the binding or uptake of 2-NBDG or 6-NBDG by L929 cells, though both approaches significantly impact [3H]-2-deoxyglucose uptake rates. Together these data indicate that 2-NBDG and 6-NBDG can bind and enter mammalian cells by transporter-independent mechanisms, which calls into question their utility as an accurate proxy for glucose transport.

2-NBDG as a marker for detecting glucose uptake in reactive astrocytes exposed to oxygen-glucose deprivation in vitro

J Mol Neurosci2015 Jan;55(1):126-130.PMID: 25091860DOI: 10.1007/s12031-014-0385-5

Glucose is a necessary source of energy for sustaining cell activities and homeostasis in the brain. Enhanced glucose uptake protects cells during energy depletion including brain ischemia. Astrocytes enhance their glucose uptake during ischemia to supply substrates to neurons and thus support neuronal survival. Radiolabeled substrates are commonly used for in vitro measurement of glucose uptake in astrocytes. Here we optimized a method to measure glucose uptake by astrocytes during oxygen-glucose deprivation (OGD) using the fluorescent substrate 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). Uptake buffers for 2-NBDG were the same as for (14)C-labeled α-methyl-D-glucopyranoside. Cell lysis buffer was optimized for observing the fluorescence of 2-NBDG, and Hoechst 33258 DNA staining was used for normalization of the 2-NBDG concentration. Uptake was performed on cultures of primary astrocytes by incubating the cells at 37 °C in buffer containing 25-200 μM 2-NBDG. Flow cytometry was performed to visualize uptake in intact cells, and a fluorescence microplate reader was used to measure the intracellular concentration of 2-NBDG in cell homogenates. 2-NBDG uptake was concentration dependent in astrocytes that were exposed or not exposed to OGD. OGD significantly increased 2-NBDG uptake by about 1.2 to 2.5 times in astrocytes compared to control cells. These results show that 2-NBDG can be used to detect glucose transport in astrocytes exposed to OGD.