An XFe96 extracellular flux analyzer was used to measure glycolysis. Cells were suspended in Seahorse XF RPMI medium containing 2 mM L-glutamine and seeded on XFe96 well microplates coated with Cell-Tak at a density of 1 × 105 cells per well. After seeding, the cells were equilibrated in a non-CO2 incubator for 20 minutes and used in the assay. After baseline measurements, glucose (10 mM), oligomycin (1 μM) and 2-deoxy-D-glucose (2-DG, 50 mM), which were adjusted using the reagents in the Seahorse XF cell glycolysis stress test kit (Agilent Technologies), were sequentially added to each well. The data are presented as the extracellular acidification rate (ECAR; mpH/minute). Glycolysis, glycolytic capacity and glycolytic reserve were calculated.
Seahorse xf cell glycolysis stress test kit
Manufactured by Agilent Technologies
Sourced in United States
The Seahorse XF Cell Glycolysis Stress Test Kit is a laboratory equipment product from Agilent Technologies. The kit is designed to measure the glycolytic function of cells in real-time. It provides data on key glycolytic parameters, including glycolytic rate, glycolytic capacity, and glycolytic reserve.
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Lab products found in correlation
Seahorse bioscience xf 96 extracellular flux analyzer, by Agilent Technologies (2 mentions)
Xf96 analyzer, by Agilent Technologies (1 mentions)
Xfe96 extracellular flux analyzer, by Agilent Technologies (1 mentions)
Xf96 extracellular flux analyzer, by Agilent Technologies (1 mentions)
Seahorse xf extracellular ux analyzer xfe96, by Agilent Technologies (1 mentions)
Seahorse xfe96 fluxpak plates, by Agilent Technologies (1 mentions)
7 protocols using seahorse xf cell glycolysis stress test kit
1
Extracellular Flux Analyzer Measures Glycolysis
2
Measuring Cellular Glycolytic Capacity
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Cells (10,000) were plated in each XF96-well microplate well (Seahorse Bioscience) followed by ECAR measurement at 37 °C using an XF96 Analyzer (Seahorse Bioscience). Then, final concentrations of 10 mM glucose, 1.5 μM oligomycin and 50 mM 2-DG from the Seahorse XF Cell Glycolysis Stress Test kit (Agilent, 103020-100) were added to each well. XF data were normalized to the protein concentration of each well, which was detected by BCA analyses.
3
Extracellular Acidification Rate Measurement
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ECAR was measured in MCF7 cells by using a Seahorse XF Cell Glycolysis Stress Test Kit (Agilent) in an Extracellular Flux Analyzer XFe96 (Seahorse Bioscience). Specifically, siRNA‐transfected or lenti‐viral‐infected MCF7 cells were re‐seeded at approximately 10000 cells per well onto a Seahorse 96‐well assay plate and incubated in XF DMEM Medium without bicarbonate, glucose, pyruvate, or glutamine (pH 7.4) (Seahorse Bioscience) in an incubator for 1 h (37 °C without CO2). Then, the pre‐warmed glucose, oligomycin, and 2‐DG were sequentially added into the injector (glucose, 10 mm ; oligomycin, 1 µm ; 2‐DG, 50 mm ). ECAR was measured three times for 6 min each, with 3 min of measuring time and 3 min of mixing time between measurements. The data obtained were used to evaluate the glycolytic function include glycolysis, glycolytic capacity, glycolytic reserve, and nonglycolytic acidification.
4
Glycolytic Capacity Measurement by ECAR
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Extracellular acidification rate (ECAR) was analyzed by the Seahorse Bioscience XF96 Extracellular Flux Analyzer (Seahorse Bioscience, USA) with Seahorse XF Cell Glycolysis Stress Test Kit (Seahorse Bioscience, USA). Briefly, 2 × 104/well HCC cells were seeded into an XF96-well plate and maintained overnight. The next day, cells were incubated with an unbuffered medium followed by sequential injection of 10 mM glucose (Glc), 0.5 μM oligomycin (Oligo), and 80 mM 2-deoxyglucose (2-DG) to measure ECAR. Finally, the ECAR value was normalized to the protein amounts of cells before making comparisons between groups.
5
Extracellular Flux Analysis of Glycolysis in HCC Cells
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The Seahorse Bioscience XF96 Extracellular Flux Analyzer (Seahorse Bioscience, USA) was used to analyze ECAR in HCC cells upon different treatments. ECAR measurement was analyzed with Seahorse XF Cell Glycolysis Stress Test Kit (Seahorse Bioscience, USA) according to the manufacturer's protocols. In this study, 10 mM glucose, 0.5-1 μM oligomycin (Oligo), and 80 mM 2-deoxyglucose (2-DG) were used for ECAR detection. The acquired data were further normalized to the corresponding protein concentration of cell extracts.
6
Extracellular Acidification Rate Analysis
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The extracellular acidification rate (ECAR) was determined using the Seahorse Bioscience XF96 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA). Experiments were performed according to the manufacturer's protocols. Seahorse XF Cell Glycolysis Stress Test Kit (Seahorse Bioscience) was used for ECAR measurement. In brief, 2 × 104 cells per well were seeded in an XF96‐well plate. After baseline measurements, glucose, the oxidative phosphorylation inhibitor oligomycin, and the glycolytic inhibitor 2‐DG were sequentially injected into each well at indicated time points. Finally, data were assessed by Seahorse XF‐96 Wave software and ECAR is shown in mpH/minute.
7
Seahorse XF Glycolysis Stress Assay
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The ECAR assays were performed using the Seahorse XF extracellular ux analyzer (XFe96) (Agilent Technologies, Santa Clara, USA) with the Seahorse XF Cell Glycolysis Stress Test Kit (103710-100, Seahorse) and Seahorse XFe96 FluxPak Plates (102416-100, Seahorse) according to the manufacturer's instructions. Brie y, 6000-8000 cells per well were seeded into the Seahorse XFp cell culture microplate overnight or treated with TAT-Pep for another 24 h. The probe plate was hydrated with deionized water for at least 4 h and then changed to calibration solution for another 1 h at 37 °C in a cell incubator without CO 2 before the assays run. The base medium (pH=7.4) was supplemented with L-glutamine (2 mM) to make the detection solution. Next, cells were washed using the detection solution and incubated in a cell incubator without CO 2 before the assays run. After baseline measurements, D-glucose (10 mM), ATP synthase inhibitor Oligomycin (1 μM), the glycolysis inhibitor 2-DG (50mM) were sequentially injected into each well at the indicated time points. Data was recorded and analyzed by the Seahorse XF Wave software. Glycolytic capacity was calculated as the difference between the average value of time point 7,8,9 and that of time point 10,11,12.
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