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2-NBDG Sale

(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是一种荧光标记的2-脱氧葡萄糖类似物,可用作细胞葡萄糖代谢评估的示踪剂(激发波长/发射波长:475/550纳米)。

2-NBDG Chemical Structure

Cas No.:186689-07-6

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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

References:
[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.

2-NBDG是一种荧光标记的2-脱氧葡萄糖类似物,可用作细胞葡萄糖代谢评估的示踪剂(激发/发射波长:475/550纳米)。

excitation and emission of 2-NBDG

葡萄糖是维持组织细胞活动和稳态所必需的能量来源。葡萄糖代谢在许多疾病中都是一个重要的靶点,并且会随着病理情况而改变,因此评估葡萄糖代谢可以成为一种重要的指标来判断疾病进展情况。

2-NBDG可以在许多种体外细胞中使用,例如HepG2人类肝癌细胞、L6大鼠骨骼肌细胞、MCF-7乳腺癌上皮细胞和星形胶质细胞,它还用于疾病模型,如癫痫大鼠、高血糖、糖尿病或小鼠异种移植的癌症模型。

2-NBDG通过葡萄糖转运体进入细胞,并被己糖激酶磷酸化并困在细胞内。可以使用流式细胞术检测细胞产生的荧光来检查2-NBDG进入活细胞的情况,也可以使用荧光微孔板分析法测量转运的2-NBDG在细胞内的浓度。它还可以简单地用荧光成像显微镜或CCD相机进行检测。

2-NBDG是一种荧光标记的葡萄糖示踪剂,通过与葡萄糖相同的葡萄糖转运体(GLUT)进入细胞。一旦2-NBDG被细胞吸收,它会在C-6位置磷酸化成为2-NBDG-6-phosphate,并且能够很好地保留在细胞内。与其他葡萄糖示踪剂如2-DG或FDG相比,2-NBDG可以以高时间和空间分辨率在单个细胞水平上原位测量2-NBDG。(适用于荧光显微镜和流式细胞术检测)

Principle of 2-NBDG

2-NBDG葡萄糖摄取实验在细胞中的原理:一旦细胞吸收了2-NBDG,它会在C-6位置被磷酸化,生成2-NBDG-6-磷酸盐,在2 NBDG代谢中很好地保留在细胞内,荧光强度与细胞内葡萄糖摄取活性成正比。

参考文献: [1]. 邹超,王勇,沈志. 2-NBDG作为直接测量葡萄糖摄取的荧光指示剂[J]. 生物化学和生物物理方法杂志, 2005, 64(3):207-215. [2]. O'Neil R G, Wu L, Mullani N. 荧光去氧葡萄糖类似物(2-NBDG)在肿瘤细胞中的摄取[J]. 分子成像与生物学, 2005,7(6):388-392. [3]. Tsytsarev V, Maslov K I,Yao J等. 使用荧光去氧葡萄糖类似物2-NBDG进行癫痫活动的体内成像[J]. 神经科学方法杂志,2012,203(1):136-140。 [4]。陈艳、张俊健、张向阳。2-NBDG作为标记探测缺氧/低血糖条件下反应性星形胶质细胞中的葡萄糖摄取[J]。分子神经科学杂志 (2015)55:126–130。 [5]。Tsytsarev V,Maslov KI,Yao J等.使用荧光去氧葡萄糖类似物2-NBDG进行癫痫活动的体内成像[J].神经科学方法杂志,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
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Research Update

2-NBDG as a fluorescent indicator for direct glucose uptake measurement

Evaluation of glucose uptake ability in cells plays a fundamental role in diabetes mellitus research. In this study, we describe a sensitive and non-radioactive assay for direct and rapid measuring glucose uptake in single, living cells. The assay is based on direct incubation of mammalian cells with a fluorescent d-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) followed by flow cytometric detection of fluorescence produced by the cells. A series of experiments were conducted to define optimal conditions for this assay. By this technique, it was found that insulin lost its physiological effects on cells in vitro meanwhile some other anti-diabetic drugs facilitated the cell glucose uptake rates with mechanisms which likely to be different from those of insulin or those that were generally accepted of each drug. Our findings show that this technology has potential for applications in both medicine and research.

Newcastle disease virus degrades SIRT3 via PINK1-PRKN-dependent mitophagy to reprogram energy metabolism in infected cells

Lacking a self-contained metabolism network, viruses have evolved multiple mechanisms for rewiring the metabolic system of their host to hijack the host's metabolic resources for replication. Newcastle disease virus (NDV) is a paramyxovirus, as an oncolytic virus currently being developed for cancer treatment. However, how NDV alters cellular metabolism is still far from fully understood. In this study, we show that NDV infection reprograms cell metabolism by increasing glucose utilization in the glycolytic pathway. Mechanistically, NDV induces mitochondrial damage, elevated mitochondrial reactive oxygen species (mROS) and ETC dysfunction. Infection of cells depletes nucleotide triphosphate levels, resulting in elevated AMP:ATP ratios, AMP-activated protein kinase (AMPK) phosphorylation, and MTOR crosstalk mediated autophagy. In a time-dependent manner, NDV shifts the balance of mitochondrial dynamics from fusion to fission. Subsequently, PINK1-PRKN-dependent mitophagy was activated, forming a ubiquitin chain with MFN2 (mitofusin 2), and molecular receptor SQSTM1/p62 recognized damaged mitochondria. We also found that NDV infection induces NAD+-dependent deacetylase SIRT3 loss via mitophagy to engender HIF1A stabilization, leading to the switch from oxidative phosphorylation (OXPHOS) to aerobic glycolysis. Overall, these studies support a model that NDV modulates host cell metabolism through PINK1-PRKN-dependent mitophagy for degrading SIRT3.Abbreviations: AMPK: AMP-activated protein kinase; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ECAR: extracellular acidification rate; hpi: hours post infection LC-MS: liquid chromatography-mass spectrometry; mito-QC: mCherry-GFP-FIS1[mt101-152]; MFN2: mitofusin 2; MMP: mitochondrial membrane potential; mROS: mitochondrial reactive oxygen species; MOI: multiplicity of infection; 2-NBDG: 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxyglucose; NDV: newcastle disease virus; OCR: oxygen consumption rate; siRNA: small interfering RNA; SIRT3: sirtuin 3; TCA: tricarboxylic acid; TCID50: tissue culture infective doses.

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

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 米m, 2.1 mm ℅ 150 mm, Waters) with acetonitrile-water containing 2 米M 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.

Characterization of a fluorescent glucose derivative 1-NBDG and its application in the identification of natural SGLT1/2 inhibitors

Glucose is an important energy source for cells. Glucose transport is mediated by two types of glucose transporters: the active sodium-coupled glucose cotransporters (SGLTs), and the passive glucose transporters (GLUTs). Development of an easy way to detect glucose uptake by the cell can be valuable for research. 1-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-1-deoxy-d-glucose (1-NBDG) is a newly synthesized fluorescent glucose analogue. Unlike 2-NBDG, which is a good substrate of GLUTs but not SGLTs, 1-NBDG can be transported by both GLUTs and SGLTs. Thus, 1-NBDG is useful for the screening of SGLT1 and SGLT2 inhibitors. Here we further characterized 1-NBDG and compared it with 2-NBDG. The fluorescence of both 1-NBDG and 2-NBDG was quenched under alkaline conditions, but only 1-NBDG fluorescence could be restored upon neutralization. HPLC analysis revealed that 2-NBDG was decomposed leading to loss of fluorescence, whereas 1-NBDG remained intact in a NaOH solution. Thus, after cellular uptake, 1-NBDG fluorescence can be detected on a plate reader simply by cell lysis in a NaOH solution followed by neutralization with an HCl solution. The fluorescence stability of 1-NBDG was stable for up to 5 h once cells were lysed; however, similar to 2-NBDG, intracellular 1-NBDG was not stable and the fluorescence diminished substantially within one hour. 1-NBDG uptake could also be detected at the single cell level and inhibition of 1-NBDG uptake by SGLT inhibitors could be detected by flow cytometry. Furthermore, 1-NBDG was successfully used in a high-throughput cell-based method to screen for potential SGLT1 and SGLT2 inhibitors. The SGLT inhibitory activities of 67 flavonoids and flavonoid glycosides purified from plants were evaluated and several selective SGLT1, selective SGLT2, as well as dual SGLT1/2 inhibitors were identified. Structure-activity relationship analysis revealed that glycosyl residues were crucial since the aglycon showed no SGLT inhibitory activities. In addition, the sugar inter-linkage and their substitution positions to the aglycon affected not only the inhibitory activities but also the selectivity toward SGLT1 and SGLT2.

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

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.