2 nbd glucose

Manufactured by Thermo Fisher Scientific

Sourced in Germany

2-NBD-glucose is a fluorescent glucose derivative that can be used to study glucose uptake and metabolism in cells. It contains a 2-(N-methylamino)benzoic acid (NBD) fluorescent tag attached to the glucose molecule, allowing for the tracking and quantification of glucose transport and utilization.

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7 protocols using 2 nbd glucose

Glucose Uptake Measurement by Flow Cytometry

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Zeng N., Okumura T., Alauddin M., Khozooei S., Rajaxavier J., Zhang S., Singh Y., Shi B., Brucker S.Y., Wallwiener D., Takeda S., Lang F, & Salker M.S. (2020). LEFTY2/endometrial bleeding-associated factor up-regulates Na+ Coupled Glucose Transporter SGLT1 expression and Glycogen Accumulation in Endometrial Cancer Cells. PLoS ONE, 15(4), e0230044.

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Insulin Signaling Pathway Modulation

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Ursolic acid, cytochalasin B, insulin, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, wortmannin, SB203580, compound C, protease inhibitor and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO, USA). High glucose Dulbecco’s modified Eagle’s medium (DMEM) was from Mediatech, Inc. (Cellgro Mediatech, Inc. Manassas, VA). Fetal bovine serum (FBS) was bought from PAA Laboratories (Etobicoke, ON, Canada). Bovine calf serum (BCS) was purchased from Cayman Chemical Company (Ann Arbor, Michigan, USA). The BCA protein assay kit was obtained from Thermo Scientific (San Jose, CA, USA). RIPA lysis buffer was from Millpore (MA, USA). Protein loading buffer was from Bio-Rad (Montreal, QC, Canada). Antibodies against phospho-phosphoinositide-dependent kinase (pPDK), phosphoinositide-dependent kinase (PDK), phospho-protein kinase C (PKC), protein kinase C (PKC), phospho-AS160 (pAS160), AS160, GLUT4, glucose transporter 1 (GLUT1), phospho-phosphoinositide-dependent serine/threonine kinase (pAKT), phosphoinositide-dependent serine/threonine kinase (AKT) and clathrin were from Cell Signaling Technology, Inc. (Beverly, Massachusetts, USA). 2-NBD-glucose was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). BM chemiluminescence blotting substrate kit was from Roche Diagnosis (Laval, QC, Canada).

He Y., Li W., Li Y., Zhang S., Wang Y, & Sun C. (2014). Ursolic Acid Increases Glucose Uptake through the PI3K Signaling Pathway in Adipocytes. PLoS ONE, 9(10), e110711.

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Glucose Uptake Measurement by 2-NBD-Glucose

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The fluorescent glucose analogue 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBD-glucose; Invitrogen, Darmstadt, Germany) was used to measure the relative uptake of glucose by flow cytometry. In each condition, cells were incubated with 2-NBD-glucose (30 μM) for 1 hour at 37 °C, subsequently washed twice in cold PBS and analyzed by flow cytometry (BD Biosciences, Heidelberg, Germany) in fluorescence channel FL1. To determine the effect of SGLT1 inhibition on glucose uptake, cells were incubated with 1 mM of the SGLT1 inhibitor, Phlorizin (1 mM; #274313; Sigma). To determine the effect of Na⁺ on total glucose uptake by SGLT1 inhibition, cells were incubated for 2 h with either HEPES or Na⁺-free Hepes. The solutions were composed of (in mM): standard Hepes: 115 NaCl, 5 KCl, 1 CaCl₂, 1.2 MgSO₄, 2 NaH₂PO₄ 10 glucose, 32.2 Hepes or sodium free Hepes: 132.8 NMDG Cl, 3 KCl, 1 CaCl₂, 1.2 MgSO₄, 2 KH₂PO₄, 32.2 Hepes, 10 mannitol, 10 glucose. All chemicals were purchased from Sigma.

Salker M.S., Singh Y., Zeng N., Chen H., Zhang S., Umbach A.T., Fakhri H., Kohlhofer U., Quintanilla-Martinez L., Durairaj R.R., Barros F.S., Vrljicak P., Ott S., Brucker S.Y., Wallwiener D., Vrhovac Madunić I., Breljak D., Sabolić I., Koepsell H., Brosens J.J, & Lang F. (2017). Loss of Endometrial Sodium Glucose Cotransporter SGLT1 is Detrimental to Embryo Survival and Fetal Growth in Pregnancy. Scientific Reports, 7, 12612.

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Insulin Signaling Pathway Modulation

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Lipopolysaccharide (Escherichia coli serotype 055:B5, LPS), compound C (purity ≥ 98 % by HPLC analysis), protein A/G PLUS-Agarose, AICAR (purity ≥ 98 % by HPLC analysis), and insulin were obtained from Sigma-Aldrich. Anti-IRS-1 (R301; BS1408); anti-Phospho-IRS-1 (Ser307; BS4725), anti-Akt (A444; BS1810), anti-Phospho-Akt (T308; BS4008), anti-IKKβ (F182; BS1407), anti-Phospho-IKKβ (Y199; BS4320), anti-Na+/K+-ATPaseα-1 (BS1436), GAPDH (AP0063), HRP-conjugated anti-rabbit, and anti-mouse IgG antibodies were purchased from Bioworld Technology. PY99 (sc-7020) was purchased from Santa Cruz Biotechnology. Histone H3, anti-AMPKα, anti-phospho-AMPKα (T172), and anti-GLUT-4 were purchased from Cell Signaling Technology. 2-NBD-glucose (N13195, lot: 873 337) was purchased from Invitrogen. Sodium salicylate (purity ≥ 99.5 %) was purchased from Tianjin Kemiou Chemical Agent Center. Fraction-PREP Cell Fractionation Kit was purchased from BioVision.

Zhao W., Ge H., Liu K., Chen X., Zhang J, & Liu B. (2017). Nandinine, a Derivative of Berberine, Inhibits Inflammation and Reduces Insulin Resistance in Adipocytes via Regulation of AMP-Kinase Activity. Planta medica, 83(3-04).

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Microencapsulated Protein Release Kinetics

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AlexaFluor488-labeled IgG (goat anti-rabbit IgG, Life Technologies), bovine serum albumin-AlexaFluor488 conjugate (Life Technologies), 2-NBD-glucose (Life Technologies) or insulin (Sigma) tagged with AlexaFluor488 was dissolved in a 5% PEG-4MAL (10 kDa or 20 kDa) solution before being microencapsulated by macromer droplet gelation. To prevent proteins from being crosslinked by the macromer, thiols were capped using aminoethylate reagent (Thermo Scientific) according to product instructions. Particles were washed and resuspended in PBS and divided into 5 replicates containing 2 mL total volume. 50 µL samples were taken of supernatant alone, as well as of supernatant containing well-mixed, protein-laden microgels. These samples were placed in a 96 well plate, and their fluorescent intensity was measured using a Perkin Elmer HTS 7000 plate reader. To generate release curves, supernatant samples were collected over the course of 3 days, and their fluorescent intensity was measured. Protein release was normalized by setting fluorescent intensity of the supernatant alone correspond to 0% protein released, and fluorescent intensity of the buffer/microgel mixture correspond to 100% protein released. This data was plotted using GraphPad Prism, and exponential best fit curves were calculated from normalized data.

Headen D.M., Aubry G., Lu H, & García A.J. (2014). Microfluidic-Based Generation of Size-Controlled, Biofunctionalized Synthetic Polymer Microgels for Cell Encapsulation. Advanced materials (Deerfield Beach, Fla.), 26(19), 3003-3008.

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Quantifying Glucose Uptake via Flow Cytometry

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To measure glucose uptake, fluorescently labeled glucose analogue was used. Cells were collected and incubated with 2-NBD glucose (Life Technologies) at 12.5 μg/ml for 30 minutes at room temperature. Cells were then washed twice with PBS and resuspended in 500 μL of PBS and analyzed in C6 Accuri Flow (excitation at 544 nm and emission at 590 nm).

Wangpaichitr M., Wu C., Li Y.Y., Nguyen D.J., Kandemir H., Shah S., Chen S., Feun L.G., Prince J.S., Kuo M.T, & Savaraj N. (2017). Exploiting ROS and metabolic differences to kill cisplatin resistant lung cancer. Oncotarget, 8(30), 49275-49292.

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Quantifying Glucose Uptake in Endothelial Cells

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ECs were seeded at a density of 2 × 10⁵ cells per well in a 6 well plate and incubated overnight. Then, ECs were incubated in serum‐free medium for 6 hours before being washed once with PBS and incubated with 100 μmol/L of 2‐(N‐(7‐Nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)Amino)‐2‐Deoxyglucose (2‐NBD‐glucose, Life Technologies, SAS) for 1 h at 37°C. Thereafter, ECs were trypsinized before being centrifuged at 300 g for 5 minutes at room temperature and washed once with PBS. Cell pellets were resuspended in 300 μl PBS and the 2‐NBD‐glucose fluorescence was determined in the FITC channel (FL‐1) using a flow cytometer (FACScan). Mean fluorescence intensity of 2‐NBD‐glucose was used to measure glucose uptake by ECs. Unstained control was used to optimize FACS settings.

Khemais‐Benkhiat S., Belcastro E., Idris‐Khodja N., Park S., Amoura L., Abbas M., Auger C., Kessler L., Mayoux E., Toti F, & Schini‐Kerth V.B. (2019). Angiotensin II‐induced redox‐sensitive SGLT1 and 2 expression promotes high glucose‐induced endothelial cell senescence. Journal of Cellular and Molecular Medicine, 24(3), 2109-2122.

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