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Purity: ≥98%
2-NBDG, an analog of 1-NBDG, is a fluorescent analogue of glucose and an indicator used for measuring glucose uptake by bacteria and live mammalian cells and in tumor biopsies. The uptake of 2-NBDG is competitively inhibited by D-glucose, but not L-glucose or sucrose, in E. coli. 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.
| Targets |
Fluorescent Dye
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|---|---|
| ln Vitro |
1. Preparation of working solution
1.1 Preparation of stock solution. To obtain 1 mM stock solution, dissolve 1 mg of 2-NBDG in 2.92 mL DDH2O. 1.2 Preparation of working solution: Dilute the stock solution with preheated serum-free cell culture medium or PBS to prepare 10-200 μ M's 2-NBDG working solution. Note: Please adjust the concentration of 2-NBDG working solution according to your specific needs. 2. Cell staining 2.1 Suspended cells: Centrifuge and collect cells, add PBS and wash twice, each time for 5 minutes. Adherent cells: Discard the culture medium and add trypsin to digest the cells. After centrifugation, discard the supernatant and wash twice with PBS for 5 minutes each time. 2.2 Add 1 mL of 2-NBDG working solution and incubate at room temperature for 5-60 minutes. 2.3 At 400 g, centrifuge at 4 ℃ for 3-4 minutes, discard the supernatant. 2.4 Add PBS to wash cells twice, each time for 5 minutes. 2.5 After resuspend cells with 1 mL of serum-free medium or PBS, observe under a microscope. If viability testing is performed, record the optical density (O.D.) at 540/570 nm using an ELISA enzyme-linked immunosorbent assay (ELISA) reader. Cell viability was calculated using a control ratio and plotted against the logarithmic concentration of the drug to calculate IC50. |
| ln Vivo |
Circulating breast cancer cells with increased uptake of fluorescent 2-NBDG were detected in mice bearing human breast cancer xenograft tumors by fluorescence imaging, suggesting clinical use of 2-NBDG as a tracer for fluorescence imaging of hypermetabolic circulating breast cancer cells [3].
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| Enzyme Assay |
Fluorescence Imaging of Circulating Breast Cancer Cells with Increased Uptake of Fluorescent 2-NBDG Starting at 1 week after implantation of tumor cells, mouse blood samples (100 μL/mouse) were collected by puncturing the mouse saphenous vein. The blood samples containing circulating breast cancer cells were incubated with 2-NBDG, a fluorescent glucose analogue purchased from Invitrogen, at a dose of 5 μg/100 μL blood, for 30 min in a dark incubator at 37 °C. Subsequently, circulating breast cancer cells were harvested with magnetic beads conjugated with anti-epithelial cell adhesion molecule (EpCAM) IgG in a protocol from the vendor, followed by fluorescence imaging of circulating breast cancer cells with cellular uptake of fluorescent 2-NBDG. Briefly, 1 μL of 1 % suspension of the magnetic beads was added to 100 μL of blood samples at the end of incubation with 2-NBDG, followed by incubation at 4 °C for 30 min with gentle shaking to facilitate binding of the magnetic beads to circulating breast cancer cells. Subsequently, the circulating breast cancer cells were collected from the blood by magnetic separation with a magnetic separation rack, washed 3 times with PBS, and transferred to a 96-well plate after re-suspending the circulating breast cancer cells in 100 μl PBS. Uptake of 2-NBDG by circulating breast cancer cells was examined under a fluorescent microscope equipped with a 488 nm filter (Olympus). Large cells with fluorescent signals derived from cellular uptake of fluorescent 2-NBDG were counted as hypermetabolic circulating breast cancer cells, in comparison to small size of normal mouse blood cells (lymphocytes and RBC) showing no or little fluorescence signals of 2-NBDG. Total number of hypermetabolic circulating breast cancer cells in a blood sample was obtained by visual scanning of the whole area of the well of the 96 well plate with manual cell counter. The experiments were repeated three times[3].
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| Cell Assay |
Staining Example 1 for 2-NBDG uptake assay: 2-NBDG may be used as a fluorescent indicator for monitoring the uptake of glucose in living cells.
1. Incubate cells with 2-NBDG (50 μM; 30 min; 37℃; 5% CO2) in glucose-free DMEM. 2. Wash cells thoroughly and measure the fluorescence by microscope. Staining Example 2 for 2-NBDG uptake assay:2-NBDG may be used as a fluorescent indicator for monitoring the uptake of glucose in living cells. 1. Incubate cells with low-glucose DMEM containing 2-NBDG (150 μg/mL; 60 min; 37℃). 2. Wash cells thoroughly and measure the fluorescence by microscope. Staining Example 3 for 2-NBDG uptake assay:2-NBDG may be used as a fluorescent indicator for monitoring the uptake of glucose in living cells. 1. Incubate cells in low-glucose DMEM with 2-NBDG (150 μg/mL; 60 min; 37℃). 2. Wash cells thoroughly and measure the fluorescence by microscope. Staining Example 4 for 2-NBDG uptake assay: 2-NBDG may be used as a fluorescent indicator for monitoring the uptake of glucose in living cells. 1. Incubate cells with 2-NBDG (100 μM; 30 min). 2. Wash cells thoroughly and measure the fluorescence by microscope. |
| Animal Protocol |
Procedures:[4]
Human breast cancer cells were implanted in the mammary gland fat pad of athymic mice to establish orthotopic human breast cancer xenografts as a mouse model of circulating breast cancer cells. Near-infrared fluorescence imaging of the tumor-bearing mice injected with 2-DeoxyGlucosone 750 (2-DG 750) was conducted to assess glucose metabolism of xenograft tumors. Following incubation with fluorescent 2-NBDG, circulating breast cancer cells in the blood samples collected from the tumor-bearing mice were collected by magnetic separation, followed by fluorescence imaging for 2-NBDG uptake by circulating breast cancer cells, and correlation of the number of hypermetabolic circulating breast cancer cells with tumor size at the time when the blood samples were collected. Results: [4] Human breast cancer xenograft tumors derived from MDA-MB-231, BT474, or SKBR-3 cells were visualized on near-infrared fluorescence imaging of the tumor-bearing mice injected with 2-DG 750. Hypermetabolic circulating breast cancer cells with increased uptake of fluorescent 2-NBDG were detected in the blood samples from tumor-bearing mice and visualized by fluorescence imaging, but not in the blood samples from normal control mice. The number of hypermetabolic circulating breast cancer cells increased along with growth of xenograft tumors, with the number of hypermetabolic circulating breast cancer cells detected in the mice bearing MDA-MB231 xenografts larger than those in the mice bearing BT474 or SKBR-3 xenograft tumors. |
| References |
[1]. Yamada K, et al. A real-time method of imaging glucose uptake in single, living mammalian cells. Nat Protoc. 2007;2(3):753-62.
[2]. Zou C, et al. 2-NBDG as a fluorescent indicator for direct glucose uptake measurement. J Biochem Biophys Methods. 2005 Sep 30;64(3):207-15. [3]. J Fluoresc. 2013 Jan;23(1):213-20. doi: 10.1007/s10895-012-1136-z. |
| Additional Infomation |
This protocol details a method for monitoring glucose uptake into single, living mammalian cells using a fluorescent D-glucose derivative, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), as a tracer. The specifically designed chamber and superfusion system for evaluating 2-NBDG uptake into cells in real time can be combined with other fluorescent methods such as Ca2+ imaging and the subsequent immunofluorescent classification of cells exhibiting divergent 2-NBDG uptake. The whole protocol, including immunocytochemistry, can be completed within 2 d (except for cell culture). The procedure for 2-NBDG synthesis is also presented.[1]
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| Molecular Formula |
C12H14N4O8
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|---|---|
| Molecular Weight |
342.26200
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| Exact Mass |
342.08117
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| Elemental Analysis |
C, 42.11; H, 4.12; N, 16.37; O, 37.40
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| CAS # |
186689-07-6
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| Related CAS # |
2376921-70-7 (1-NBDG)
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| PubChem CID |
6711157
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| Appearance |
Typically exists as light yellow to orange solids at room temperature
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| Density |
1.750±0.06 g/cm3
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| Boiling Point |
707.6±70.0 °C at 760 mmHg
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| Flash Point |
381.7±35.7 °C
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| Vapour Pressure |
0.0±2.4 mmHg at 25°C
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| Index of Refraction |
1.77
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| LogP |
-0.41
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| tPSA |
186.92
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| SMILES |
O=C[C@H](NC1=CC=C([N+]([O-])=O)C2=NON=C21)[C@H]([C@@H]([C@@H](CO)O)O)O
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| InChi Key |
QUTFFEUUGHUPQC-ILWYWAAHSA-N
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| InChi Code |
InChI=1S/C12H14N4O8/c17-3-6(11(20)12(21)8(19)4-18)13-5-1-2-7(16(22)23)10-9(5)14-24-15-10/h1-3,6,8,11-13,18-21H,4H2/t6-,8+,11+,12+/m0/s1
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| Chemical Name |
2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose
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| Synonyms |
2-NBDG; 2NBDG; 2 NBDG
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
H2O : ~5 mg/mL (~14.61 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: 3.33 mg/mL (9.73 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).
(Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9218 mL | 14.6088 mL | 29.2176 mL | |
| 5 mM | 0.5844 mL | 2.9218 mL | 5.8435 mL | |
| 10 mM | 0.2922 mL | 1.4609 mL | 2.9218 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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