Cellular hexokinase activity was measured utilizing the Glucose Uptake-Glo™ Assay kit (Promega) with a modification from the protocol described above. Cells were treated with or without PDGF for 10 min, and rinsed with glucose-free DMEM and PBS. Cells were then incubated in reaction buffer (50 mM Tris–HCl pH 7.4, 10 mM MgCl2, 1% Triton X-100, 1 mM 2DG) containing the indicated concentration of ATP, instead of 2DG-containing PBS, at 37°C for 10 min. The reactions were terminated by adding Stop Buffer from the kit, and then the mixtures were neutralized. The mixtures were then mixed with the 2DG6P detection reagent and incubated at RT for 1 h. Luminescence signals were quantified using SpectraMaxiD5 plate reader (Molecular Devices). Absolute amounts of 2DG6P production were calculated based on signals of 2DG6P standards. 2DG6P production was normalized by amounts of protein per well.
Glucose uptake glo assay kit
Manufactured by Promega
Sourced in United States
The Glucose Uptake-Glo Assay kit is a bioluminescent assay designed to measure glucose uptake in cells. The kit utilizes a glucose detection reagent to quantify the amount of glucose taken up by cells, providing a sensitive and reliable method for analyzing cellular glucose metabolism.
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Lab products found in correlation
Humalog insulin, by Eli Lilly (2 mentions)
2 deoxyglucose, by Merck Group (2 mentions)
Lactate glo assay kit, by Promega (2 mentions)
A1443001, by Thermo Fisher Scientific (2 mentions)
Synergy ht, by Agilent Technologies (2 mentions)
Spectramax id5, by Molecular Devices (1 mentions)
Cl316 243, by Bio-Techne (1 mentions)
Spironolactone, by Merck Group (1 mentions)
Forskolin, by Bio-Techne (1 mentions)
Suramin, by Bio-Techne (1 mentions)
Carbenoxolone, by Merck Group (1 mentions)
Silencer select sirna, by Thermo Fisher Scientific (1 mentions)
Arl 67156 trisodium salt, by Bio-Techne (1 mentions)
8 br camp, by Bio-Techne (1 mentions)
Nad nadh glo assay kit, by Promega (1 mentions)
Atplite assay kit, by PerkinElmer (1 mentions)
Ros glo assay kit, by Promega (1 mentions)
Tmre mitochondrial membrane potential assay kit, by Abcam (1 mentions)
2 nbdg, by Cayman Chemical (1 mentions)
Spectramax i3x, by Molecular Devices (1 mentions)
48 protocols using glucose uptake glo assay kit
1
Measuring Cellular Hexokinase Activity
2
Insulin-stimulated Glucose Uptake Assay
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Mice were fasted for 5 h, then injected i. p. with Humalog insulin (Lilly; 0.75 U/kg body weight), followed 10 min later with an injection of 2-deoxyglucose (Sigma–Aldrich; 32.8 μg/g body weight). Tissues were collected 20 min after administration of 2-deoxyglucose. Tissues were lysed in 10 mM Tris–HCL, pH 8.0, by boiling for 15 min 2-deoxyglucose-6-phosphate (2DGP) was measured using a Glucose Uptake-Glo Assay Kit (Promega) following the manufacturer's instructions.
3
Dissecting Metabolic Pathways through Pharmacological Modulation
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CL316243, forskolin, 8Br-cAMP, ARL 67156 trisodium salt, and suramin were procured from Tocris (Minneapolis, MN). Carbenoxolone, trovofloxacin, and spironolactone were from Sigma Aldrich (St. Louis, MO). Silencer select siRNAs against mouse Panx1 (assay ID-s79985) or mouse Gβ subunits (GNB1 – assay ID –ss66813; GNB2 – assay ID –ss66816; GNB3 – assay ID –ss66822; GNB4 – assay ID –n420113) and silencer select negative control-2 siRNA (4390846) were purchased from ThermoFisher Scientific (Middletown, VA). Antibodies used in the study were as follows: mouse monoclonal anti-Panx1 (Clone 720505, #MAB7097) was from R&D Systems (Minneapolis, MN); rabbit-polyclonal anti-UCP1 (#U6382) was from Sigma–Aldrich (St. Louis, MO); mouse monoclonal OXPHOS antibody cocktail (#MS604) was from Mitosciences (Eugene, OR); rabbit monoclonal anti-Panx1 antibody (Clone D9M1C, #91137), anti-PKC phosphorylation antibody sampler kit (#9921) and rabbit monoclonal anti-vinculin (Clone E1E9 V, #13901) were from Cell Signaling (Danver, MA). Cellular glucose uptake was measured using a glucose uptake-Glo assay kit (#J1341, Promega, Madison, WI).
4
Quantifying 2-DG Uptake in Cells
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Cells seeded in a 96-well plate (2 × 104 cells/well) were incubated with 1 mM 2-DG for 10 min. 2-deoxyglucose-6-phosphate (2-DGP) was determined using Glucose uptake-Glo assay kit (Promega, Madison, WI, USA) following the manufacturer's instruction.
5
Metabolic Profiling of Cell Cultures
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MMP was investigated using the TMRE Mitochondrial Membrane Potential Assay Kit (Abcam). Intracellular ATP level at steady state was measured using ATPlite Assay Kit (PerkinElmer). Lactate level was measured using Lactate-Glo Assay Kit (Promega). Glucose uptake was assessed using Glucose Uptake-Glo Assay Kit (Promega). NAD+ and NADH levels were measured using NAD/NADH-Glo Assay Kit (Promega). ROS level was measured using ROS-Glo Assay Kit (Promega). The assays were performed according to the manufacturer's instructions. See supplemental information for detailed procedures.
6
Glucose Uptake Measurement Protocols
7
Glucose Uptake Quantification Assay
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Glucose uptake assay was measured with 96-well low adherent white luminescent plates. Prior to the assay, the culture medium was removed and the cells were washed with 100 μL of phosphate-buffered saline (PBS). To initiate glucose uptake, 50 μL of 2-Deoxy-d -Glucose (2DG, 1 mmol/L) in PBS was added to cells for 60 min. The uptake reaction was then stopped and samples were processed as described in the standard protocol of the Glucose Uptake Glo Assay kit (Promega, Madison, WI, USA). Luminescence was read with 0.3–1 s integration on a luminometer (Thermo Fisher Scientific, Scotland, UK) and the rate of glucose uptake was expressed as fmol/min/cell.
8
Glucose Uptake Quantification via 2-DG Assay
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Glucose Uptake was measured using the Glucose Uptake-Glo Assay kit (Promega Cat# J1341). Cells were incubated in a tissue culture incubator (37 °C, 5% CO2), and were treated with CR-31. After 5 h of treatment, the medium was replaced with PBS, and treated with 1 mM 2-deoxyglucose for 10 min. Stop Buffer, Neutralization Buffer, and 2DG6P Detection Reagent provided by the assay kit were subsequently added, incubated at room temperature for 30 min. Luminescence was read on a luminometer (Spectramax i3x, Molecular Devices).
9
Glycolytic Metabolic Profiling of Cancer Cells
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Methods for measuring glycolytic metabolic alterations of cellular metabolism have been described in our previous study (20 (link)). In short, 1 × 104 KYSE30 and KYSE150 cells were inoculated into 96-well plates and treated with sulconazole (30 μM and 50 μM) for 24 h. Then, glucose uptake and lactate production were measured with a Glucose Uptake-Glo Assay kit (Promega).
10
Glucose Uptake Assay with RXC004
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SNU-1411, HPAF-II, and HCT116 cells were treated in vitro for 48 hours with RXC004 (100 nmol/L), Latrunculin A (Abcam #ab144290; 500 nmol/L), or DMSO prior to analysis using the Glucose Uptake Glo Assay Kit (Promega #J1341). The ATPlite assay was used to control for cell viability, confirming RXC004 has no effect on proliferation at this 48 hours timepoint.
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