Glucose uptake was performed by detecting the uptake of 2-NBDG by culture cells using the 2-NBDG Glucose Uptake Assay Kit (Abcam) according to the manufacturer’s instructions. In brief, cells were incubated with glucose-free HBSS medium for 30 minutes after washing with PBS. After washing with PBS, cells were incubated with 2-NBDG (100 μM) for 30 minutes at 37°C. Cells were then resuspended in 150 μL of prechilled PBS containing 0.5% BSA, and flow cytometry analysis was performed within 30 minutes.
2 nbdg glucose uptake assay kit
Manufactured by Abcam
Sourced in United Kingdom, United States
The 2-NBDG glucose uptake assay kit is a fluorescence-based tool for the quantitative measurement of glucose uptake in cells. It utilizes the fluorescent glucose analog 2-NBDG to monitor glucose uptake activity. The kit provides all the necessary reagents and components to perform the assay.
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Lab products found in correlation
Lactate production assay kit, by Abcam (2 mentions)
Novocyte flow cytometer, by Agilent Technologies (1 mentions)
Anhydrous pyridine, by Merck Group (1 mentions)
Methoxamine hydrochloride, by Merck Group (1 mentions)
Rosiglitazone, by Merck Group (1 mentions)
ρ nitrophenyl ρ d glucopyranosidase, by Merck Group (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Quercetin, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
N hexane, by Merck Group (1 mentions)
Methylene blue, by Merck Group (1 mentions)
3t3 l1 cell line, by American Type Culture Collection (1 mentions)
Atp assay kit, by Abcam (1 mentions)
Mitochondrial ros detection assay kit, by Cayman Chemical (1 mentions)
Ros assay kit, by Beyotime (1 mentions)
19 protocols using 2 nbdg glucose uptake assay kit
1
Quantitative Glucose Uptake Assay
2
Fluorescent Glucose Uptake Assay
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The glucose uptake was measured using the 2-NBDG Glucose Uptake Assay Kit (Abcam, Cambridge, UK) following the manufacturer’s recommendations. Briefly, after 1 h of glucose deprivation, the cells were incubated with a glucose-free medium containing a fluorescently-labeled deoxyglucose analog, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG, 100 µg/mL), for 45 min at 37 °C. The cells were washed with a Cell-Based Assay Buffer twice to remove the excess 2-NBDG and then detected by a NovoCyte Flow Cytometer (ACEA Biosciences, San Diego, CA, USA), using a filter with an excitation/emission = 485/535 nm.
3
Evaluation of Antidiabetic Compounds in 3T3-L1 Cells
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The organic chemicals of analytical grade, namely ethanol, methanol, n-hexane, ethyl acetate, and dimethyl sulfoxide (DMSO), were obtained from Merck (Darmstadt, Germany). The four inorganic salts (sodium chloride, potassium chloride, calcium chloride, and magnesium sulfate) and methylene blue were purchased from Merck (Darmstadt, Germany). Ascorbic acid, rosiglitazone, ρ-nitrophenyl-ρ-D-glucopyranosidase (PNPG), quercetin, insulin, pyridine anhydrous, methoxamine hydrochloride, N-methyl-N-(trimethylsilyl) tryfluoroacetamide (MSTFA), and phosphate-buffered saline (PBS) were bought from Sigma-Aldrich (St. Louis, MO, USA). The AG enzyme (source: yeast maltase) was purchased from Megazyme, Ireland. Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco, Life Technologies (Carlsbad, CA, USA). Furthermore, the 2-NBDG Glucose Uptake assay kit and tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were procured from Abcam (Cambridge, UK) and Life Technologies (Carlsbad, CA, USA), respectively. The 3T3-L1 cell line was obtained from American Type Culture Collection ATCC (Manassas, VA, USA).
4
Apatinib Modulates Glucose Uptake
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5 × 105 A549 or H460 cells per well were cultured and treated with apatinib. The cells were harvested and incubation in 0.1 mM 2-NBDG (2-NBDG Glucose Uptake Assay Kit; Abcam, Cambridge, UK) for 15 min. Then the supernatant was discharged and the cells were rinsed two times with PBS and then collected for flow cytometry.
5
Extracellular Lactate and Glucose Uptake
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Extracellular lactate levels and glucose uptake were measured using the Lactate production assay kit (Biovision#K607-100) and 2-NBDG glucose uptake assay kit (Abcam#ab235976), respectively, according to the manufacturer’s instructions.
6
Glucose Uptake Assay in Hepatocytes
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Samples were processed as detailed in the manual of the 2-NBDG Glucose Uptake Assay Kit (Abcam). Briefly, hepatocytes were plated in EMEM medium in 6-well plates reaching about 80% confluency, and then the cells were serum-starved for 6 h. Following starvation, replace the serum-free medium with pre-warmed glucose-free DMEM, and hepatocytes were treated with ATP or UTP for 30 min in total, during which the final 10 min of the cells were incubated with 2-NBDG at the final concentration of 200 μg/mL. After NBDG was taken up by the cells, fluorescence was determined immediately with an excitation wavelength set to 485 nm and an emission wavelength set to 535 nm.
7
Metabolic Profiling of HepG2 Cells
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Cellular ATP, ROS, extracellular lactate levels, mitochondrial ROS, and glucose uptake were measured using the ATP assay Kit (Biovision), ROS assay kit (Beyotime), Lactate production assay kit (Biovision), Mitochondrial ROS Detection Assay kit (Cayman Chemical), and 2‐NBDG glucose uptake assay kit (Abcam), respectively, according to the manufacturer's instructions. Fru‐2,6‐BP levels in HepG2 DDIT3‐WT and ‐KO cells treated with or without glutamine were determined by Quantitative Metabolomics based on LC‐MS from BGI (The Beijing Genomics Institute) company, Shenzhen, China.
8
Fluorescence-Based Glucose Uptake Assay
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The 2-NBDG glucose uptake assay kit (ab235976, Abcam, Cambridge, UK) employs a fluorescent labeled deoxyglucose analog that can be used for the detection of glucose uptake by cultured cells. Cells were seeded in a 96-well plate with 25 000 cells per well. After 72 h of adherence, cell medium was replaced for 2 h with glucose-free RPMI medium. Afterwards, cells were incubated with indicated compounds for 30 min. Followed by a 15 min incubation with Hoechst 33342 (10 μg/mL, Sigma-Aldrich). 2-NBDG mix was prepared by a 50-fold dilution in glucose-free medium. 2-NBDG mix (50 μL) was added to each well for 10 min at 37°C. Afterwards, the plate was centrifuged for 1 min at 200 g. The supernatant was replaced and 50 μL cell-based assay buffer was added. Fluorescence was read at 50 ms integration time using the ImageXpress Pico Automated Cell Imaging System and quantified with the CellReporterXpress Image Acquisition and Analysis Software. All cell average FITC intensities were normalized against nuclei staining and against the control condition.
9
Metformin Enhances Glucose Uptake in Breast Cancer Cells
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Glucose uptake was measured using a 2‐deoxy‐2‐[(7‐nitro‐2,1,3‐benzoxadiazol‐4‐yl)amino]‐d ‐glucose (2‐NBDG) glucose uptake assay kit (K682, BioVision) following a previous report.27 (link) MDA‐MB‐231(BR)‐Luc cells were seeded in a 6 cm dish at a density of 2 × 105 cells. Cells were treated with 5 mM metformin for 4 h, exposed to 10 Gy X‐rays, and harvested after 24 h using trypsin‐EDTA. Cells were incubated with 2‐NBDG for 1 h in DMEM containing 0.5% FBS in a CO2 incubator, washed, and suspended using an analysis buffer. Flow cytometry was performed on a BD Biosciences flow cytometer and analyzed with FlowJo.
10
Glucose Uptake Measurement in MDMs
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Glucose uptake was measured using 2-NBDG Glucose Uptake Assay Kit (BioVision) according to the manufacturer’s instructions. Briefly, MDMs (105 cells) were seeded in a 24-well plate with 400 μl detection reagent (376 μl 0.5% serum 1,640 cell culture medium, 4 μl 2-NBDG reagent, and 20 μl glucose uptake enhancer) at 37°C for 30 min. Subsequently, cells were washed with iced analysis buffer three times and scraped off with rubber policeman. Fluorescence intensity was examined by a Beckman flow cytometer (excitation at 488 nm).
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