The cells were incubated with the peptides when confluence was reached for the indicated time. Then, they were incubated for 30 min in glucose-free medium (RPMI, Sigma-Aldrich) and subsequently for 1 h in glucose-free medium supplemented with 146 μM of the fluorescent glucose analogue 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]−2-deoxy-d -glucose), which comprises a glucose moiety with an N-nitrobenzoxadiazole (NBD)-amino group (fluorophore) at carbon 2 replacing the hydroxyl group, or 6-NBDG (6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]−6-deoxy-d -glucose), whose NBD-amino group is placed at carbon 6 and can therefore not be phosphorylated by HKs [57] (link) (Thermo Fisher). The cells were then washed with ice-cold PBS and lysed, scraped, and homogenised by 10 passages through a 25-gauge needle. Homogenates were centrifuged and the fluorescence of supernatants was measured in a microplate reader (Appliskan; Thermo Electron Corporation, Thermo Scientific). For 2-NBDG uptake, a standard curve was generated by measuring the fluorescence of a range of 2-NBDG concentrations in lysis buffer (1% Nonidet P-40, 1% sodium deoxycholate, 40 mM KCl, and 20 mM Trizma Base [Sigma-Aldrich], pH 7.4). 2-NBDG and 6-NBDG uptake was normalised to protein content of the samples.
2 nbdg
Manufactured by Merck Group
Sourced in United States, Germany
2-NBDG is a fluorescent glucose analog that can be used to measure glucose uptake in cells. It is a non-radioactive, cell-permeable 2-deoxyglucose derivative that is labeled with the fluorescent dye 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose.
Automatically generated - may contain errors
Lab products found in correlation
2 nbdg, by Thermo Fisher Scientific (8 mentions)
Mitotracker red, by Thermo Fisher Scientific (4 mentions)
Insulin, by Merck Group (3 mentions)
Metformin, by Merck Group (3 mentions)
Anti phospho ampk thr172, by Cell Signaling Technology (2 mentions)
Rabbit anti gapdh antibody, by Abcam (2 mentions)
Anti ampk thr172, by Cell Signaling Technology (2 mentions)
Uric acid, by Merck Group (2 mentions)
N 7 nitrobenz 2 oxa 1 3diazol 4 yl amino 2 deoxy d glucose 2 nbdg, by Thermo Fisher Scientific (2 mentions)
Anti phospho irs1 ser307, by Thermo Fisher Scientific (2 mentions)
5 amino 4imidazole 1 β d carboxamide ribofuranoside aicar, by MedChemExpress (2 mentions)
Anti akt ser473, by Bioworld Technology (2 mentions)
Anti phospho aktser473, by Bioworld Technology (2 mentions)
Anti irs1 ser307, by Thermo Fisher Scientific (2 mentions)
Compound c, by Merck Group (2 mentions)
Sucrose, by Merck Group (2 mentions)
Glucose, by Merck Group (2 mentions)
Facsdiva 6, by BD (2 mentions)
Accutrend lactate analyzer, by Nova Biomedical (2 mentions)
L glutamine, by Merck Group (2 mentions)
58 protocols using 2 nbdg
1
Measuring Glucose Uptake in Cells
2
Hypoxia and pH Sensing in 3D Cell Culture
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U-251 MG cells were stained with the CytoTell Red dye prior to loading and embedded in a 4 mg/mL collagen solution. After 48 h of culture in the device, the medium in the side channels was replaced with a solution of 5 µM Image IT Hypoxia Green combined with propidium iodide (PI; Sigma-Aldrich) at a final concentration of 10 μg/mL. For pH characterization, the medium in the microdevice was exchanged with medium containing a dual-labeled pH indicator dextran (70,000 MW; labeled with fluorescein and tetramethylrhodamine (TAMRA) Sigma-Aldrich) at a concentration of 100 µg/mL. The microfluidic devices were incubated for 2 h at 37 °C before microscopy imaging. The ratio of fluorescence intensities of fluorescein and TAMRA was converted to pH values using a calibration curve based on the linear portion of the pH-dependency of the emission intensity of fluorescein in a diluted aqueous solution [49 (link)]. The TAMRA in the dual-labeled dextran is used as a reference dye with a pH-insensitive fluorescence quantum yield. For evaluation of the delivery and uptake of the fluorescent glucose analogue 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG; Sigma-Aldrich), the medium in the side channels was replaced with a 200 µM 2-NBDG. The surface of the devices was also covered with the same solution and incubated for 90 min at 37 °C until microscopy imaging.
3
Isolation and Functional Analysis of Neutrophils
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PMN were isolated from heparinized blood via dextran density sedimentation and hypotonic lysis of red blood cells as described [51 (link)]. Cells were routinely >95% pure and >99% viable. For apoptosis assays, PMN were stimulated for 18 hr in RPMI-1640 with 10% human serum and TNF-α (20ng/ml), LPS (0.5μg/ml), or Pam3CSK4 (5μg/ml) as indicated before labeling with Annexin V and propidium idodide and detection using LSRII. For quantitation of glucose accumulation, PMN were stimulated as above for 2 hours with 500 μM 2-NBDG (Sigma) added concurrently with stimulation; 2-NBDG levels in CD15+ cells were detected using LSRII. Supernatants from stimulated PMN incubations were stored frozen and cytokines were quantified by batch analysis enzyme-linked immunosorbent assays (ELISA) (OptEIA ELISA kit; BD Biosciences, CA).
4
Glucose Uptake Measurement in hESC-ECs
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Glucose uptake was measured using the fluorescence-labeled deoxyglucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG, Apex Bio, USA). After the indicated treatment, hESC-ECs were incubated with 2-NBDG for 1 h at 37 °C, and the fluorescence intensity was measured using the Guava EasyCyte™ 8 flow cytometer (EMD Millipore, Germany).
5
Quantification of Cellular Glucose Uptake
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Glucose uptake activity was measured by a fluorescent D‐glucose analogue 2‐(N‐(7‐nitrobenz‐2‐oxa‐1,3‐diaz‐ol‐4‐yl) amino)‐2‐deoxy‐d ‐glucose (2‐NBDG; Thermofisher, Waltham, MA, USA) assay. Briefly, 1 × 106 cells were collected and washed, then incubated with 200 μmol/L 2‐NBDG for 1 hour at 37°C in a humidified incubator with 5% CO2. After incubation, the mean fluorescence intensity (MFI) of intracellular 2‐NBDG was immediately measured using a ImageStreamX Mark II imaging flow cytometer on FITC channel (Merck, Darmstadt, Germany).
6
Lipid Membrane Composition and Labeling
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1-Palmitoyl-2-oleoylglycero-3-phosphocholine
(POPC), egg sphingomyelin (SM), cholesterol (Ch), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)(ammonium salt) (Rho-DOPE) were
purchased from Avanti Polar Lipids (Alabaster, AL). Poly(1,2-butadiene)-b-poly(ethylene oxide) (PBD22-b-PEO14) with the average molecular weights of 1200 and
600 g/mol for the PBD and PEO blocks, respectively, was purchased
from Polymer Source Inc. (Dorval, Quebec, Canada). Sucrose, glucose,
and chloroform were purchased from Sigma-Aldrich (St. Louis, MO). N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium
salt (NBD-PE) and 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose
(2-NBDG) were purchased from ThermoFisher Scientific (Eugene, OR).
Sucrose, glucose, and 2-NBDG solutions were prepared in deionized
water (DI) (Millipore, Sigma, St. Louis, MO) were used for experimentation.
(POPC), egg sphingomyelin (SM), cholesterol (Ch), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)(ammonium salt) (Rho-DOPE) were
purchased from Avanti Polar Lipids (Alabaster, AL). Poly(1,2-butadiene)-b-poly(ethylene oxide) (PBD22-b-PEO14) with the average molecular weights of 1200 and
600 g/mol for the PBD and PEO blocks, respectively, was purchased
from Polymer Source Inc. (Dorval, Quebec, Canada). Sucrose, glucose,
and chloroform were purchased from Sigma-Aldrich (St. Louis, MO). N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium
salt (NBD-PE) and 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose
(2-NBDG) were purchased from ThermoFisher Scientific (Eugene, OR).
Sucrose, glucose, and 2-NBDG solutions were prepared in deionized
water (DI) (Millipore, Sigma, St. Louis, MO) were used for experimentation.
7
Metabolic Regulation in Cardiomyocytes
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Uric acid (15 mg/dl, nal concentration), metformin, insulin (100 nM, nal concentration) and compound C (20 µM, nal concentration) were purchased from Sigma (St. Louis, MO, USA). N-(7-Nitrobenz-2-oxa-1,3diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG, 100 µM, nal concentration) and anti-IRS1 (Ser307) and anti-phospho-IRS1 (Ser307) antibodies were purchased from Invitrogen (Carlsbad, CA, USA). 5-Amino-4imidazole-1-β-D-carboxamide ribofuranoside (AICAR, 500 µM, nal concentration) was purchased from MedChemExpress (MCE LLC, USA). A rabbit anti-GAPDH antibody was purchased from Abcam (Abcam, USA). Anti-Akt (Ser473) and anti-phospho-Akt (Ser473) antibodies were purchased from Bioworld (St. Louis Park, USA). Anti-AMPK (Thr172) and anti-phospho-AMPK (Thr172) antibodies were purchased from Cell Signaling Technology (CST, Beverly, MA, USA). Cardiomyocytes were cultured in serum-free media for 24 hours and then incubated in the presence of 15 mg/dl HUA for 24 hours. Cardiomyocytes were pretreated with either metformin (0 to 20 µmol/L) or AICAR, an AMPK activator (500 µmol/L), for 60 minutes before the addition of HUA. Other cells were preincubated with compound C for 6 hours before the addition of either metformin or AICAR. Then, 2-NBDG uptake was analyzed in cardiomyocytes.
were from Millipore (Billerica, MA).
were from Millipore (Billerica, MA).
8
Glucose Uptake in Cardiomyocytes
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Uric acid (15 mg/dl, nal concentration), metformin, insulin (100 nM, nal concentration) and compound C (20 µM, nal concentration) were purchased from Sigma (St. Louis, MO, USA). N-(7-Nitrobenz-2-oxa-1,3diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG, 100 µM, nal concentration) and anti-IRS1 (Ser307) and anti-phospho-IRS1 (Ser307) antibodies were purchased from Invitrogen (Carlsbad, CA, USA). 5-Amino-4imidazole-1-β-D-carboxamide ribofuranoside (AICAR, 500 µM, nal concentration) was purchased from MedChemExpress (MCE LLC, USA). A rabbit anti-GAPDH antibody was purchased from Abcam (Abcam, USA). Anti-Akt (Ser473) and anti-phospho-Akt (Ser473) antibodies were purchased from Bioworld (St.
Louis Park, USA). Anti-AMPK (Thr172) and anti-phospho-AMPK (Thr172) antibodies were purchased from Cell Signaling Technology (CST, Beverly, MA, USA). Cardiomyocytes were cultured in serum-free media for 24 hours and then incubated in the presence of 15 mg/dl HUA for 24 hours. Cardiomyocytes were pretreated with either metformin (0 to 20 µmol/L) or AICAR, an AMPK activator (500 µmol/L), for 60 minutes before the addition of HUA. Other cells were preincubated with compound C for 6 hours before the addition of either metformin or AICAR. Then, 2-NBDG uptake was analyzed in cardiomyocytes.
were from Millipore (Billerica, MA).
Louis Park, USA). Anti-AMPK (Thr172) and anti-phospho-AMPK (Thr172) antibodies were purchased from Cell Signaling Technology (CST, Beverly, MA, USA). Cardiomyocytes were cultured in serum-free media for 24 hours and then incubated in the presence of 15 mg/dl HUA for 24 hours. Cardiomyocytes were pretreated with either metformin (0 to 20 µmol/L) or AICAR, an AMPK activator (500 µmol/L), for 60 minutes before the addition of HUA. Other cells were preincubated with compound C for 6 hours before the addition of either metformin or AICAR. Then, 2-NBDG uptake was analyzed in cardiomyocytes.
were from Millipore (Billerica, MA).
9
Quantifying Cellular Glucose Uptake and Lactate Production
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For glucose uptake assays, cells were cultured in 60 cm plates, and were starved for 24 hrs with serum-free and glucose-free DMEM, then subjected to 20 μM 2-NBDG (Sigma 72987) containing glucose-free DMEM for different time points. Last, the cellular glucose uptake was quantified by FACS analysis using BD FACSDiva 6.1.3 software.
For lactate production assays, cells were planted into 60 cm plates, and were cultured for 24 hrs, then the cultured medium was collected, and lactate concentration was measured with lactate test strips and Accutrend Lactate analyzer (Accutrend Lactate, Nova Biomedical). At the same time, the viable cell numbers were calculated under the microscope. Finally, the relative lactate production was calculated with the formula (relative lactate production = lactate concentration/cell numbers) and normalized with the ratio of control cells.
For lactate production assays, cells were planted into 60 cm plates, and were cultured for 24 hrs, then the cultured medium was collected, and lactate concentration was measured with lactate test strips and Accutrend Lactate analyzer (Accutrend Lactate, Nova Biomedical). At the same time, the viable cell numbers were calculated under the microscope. Finally, the relative lactate production was calculated with the formula (relative lactate production = lactate concentration/cell numbers) and normalized with the ratio of control cells.
10
Intraperitoneal Treatments for Zebrafish
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For pharmacological treatments via intraperitoneal injections (IP), fish were randomized and subjected to IP injections at the designated time-points, with either 2DG (Sigma-Aldrich, 0.5 mg/g diluted in 1 x Phosphate Buffered Saline (PBS)), S.O. (Sigma-Aldrich, 0.6 mg/g diluted in 1 x PBS), UK-5099 (Sigma-Aldrich, 0.02 mg/g, diluted in a mixture of 1 x PBS and DMSO (1:1)) or with corresponding vehicle (Control). IP injections were performed with an insulin syringe U-100 G 0.3 mL and a 30 G needle (BD Micro-fine) inserted close to the pelvic girdle. For 3PO (Sigma-Aldrich) and MB-6 (Calbiochem) treatments, compounds were diluted in DMSO and added to water from the circulating system to a final concentration of 15 µM and 2.5 µM, respectively, and controls with equivalent amount of vehicle. For all experiments water was replaced daily and fish left to regenerate until the desired time-point.
For glucose uptake assay, fish were administered with the glucose analogue 2-NBDG (Sigma-Aldrich, 25 µmol/kg, from stock solution dissolved in DMSO) via IP injection 1 hr prior to imaging. For S-phase labeling, fish were subjected to caudal fin amputation and administrated with Ethynyl-2´-deoxyuridine (EdU, Thermo Scientific: C10337, 20 µL of 10 mM solution diluted in 1 x PBS) via IP injection 3 hr prior to caudal fin collection.
For glucose uptake assay, fish were administered with the glucose analogue 2-NBDG (Sigma-Aldrich, 25 µmol/kg, from stock solution dissolved in DMSO) via IP injection 1 hr prior to imaging. For S-phase labeling, fish were subjected to caudal fin amputation and administrated with Ethynyl-2´-deoxyuridine (EdU, Thermo Scientific: C10337, 20 µL of 10 mM solution diluted in 1 x PBS) via IP injection 3 hr prior to caudal fin collection.
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