Primary astrocytes were plated at a density of 2,000 cells/well in XF96 plate. The procedures of the glycolytic rate assay were conducted according to the user guide of Agilent Seahorse XF Glycolytic Rate Assay Kit (103344-100, Agilent Technologies). The day before assay, assay medium was prepared by supplementing base medium with 10 mM glucose, 1 mM pyruvate, 2 mM glutamine and 5.0 mM HEPES (pH 7.4). After 72 h stimulation with corticosterone and fluoxetine, cells were washed with warm assay medium for two times and the XF96 cell culture plate was calibrated in a non-CO2 incubator at 37°C for 1 h. After calibration, OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) were measured by seahorse analyzer. Rotenone/antimycin A (0.5 μM) and 2-DG (5 mM) were added to detect the compensatory glycolysis and post 2-DG acidification. GlycoPER (glycolytic proton efflux rate) was calculated according to the values of OCR and ECAR. After seahorse glycolytic rate assay, the DNA level per cell was detected by commercial kit (C7026, Invitrogen). The results of glycoPER were normalized by cell content (the ratio of DNA level).
Agilent seahorse xf glycolytic rate assay kit
Manufactured by Agilent Technologies
The Agilent Seahorse XF Glycolytic Rate Assay Kit is a laboratory equipment product designed to measure the glycolytic rate of cells. It provides quantitative data on the extracellular acidification rate (ECAR) of cells, which is an indicator of their glycolytic activity.
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Lab products found in correlation
Xf96 extracellular flux analyzer, by Agilent Technologies (2 mentions)
Seahorse xfe, by Agilent Technologies (2 mentions)
Xf calibrant solution, by Agilent Technologies (2 mentions)
Xfe24 extracellular flux analyzer, by Agilent Technologies (2 mentions)
C7026, by Thermo Fisher Scientific (1 mentions)
Agilent seahorse xf cell energy phenotype test kit, by Agilent Technologies (1 mentions)
Xf cell mito stress test kit, by Agilent Technologies (1 mentions)
Xf24 analyzer, by Agilent Technologies (1 mentions)
Seahorse xfe24 flux analyzer, by Agilent Technologies (1 mentions)
Xf96 cell culture microplate, by Agilent Technologies (1 mentions)
Bca kit, by Beyotime (1 mentions)
Seahorse xfe96 analyzer, by Agilent Technologies (1 mentions)
8 protocols using agilent seahorse xf glycolytic rate assay kit
1
Glycolytic Rate Assay in Astrocytes
2
Extracellular Flux Analysis of Cell Metabolism
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The OCR and ECAR of cells were measured with an XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA). Cells were plated (10,000 cells per well for MIA PaCa-2; 5000 cells per well for PANC-1) in at least triplicate for each condition the day before the experiment. The energy phenotype test and glycolytic rate assay were performed as described in the user guides for the Agilent Seahorse XF Cell Energy Phenotype Test Kit (103325–100, Agilent Technologies) and the Agilent Seahorse XF Glycolytic Rate Assay Kit (103344–100, Agilent Technologies). OCR and ECAR were normalized to the cell number as determined by CellTiter-Glo analysis at the end of the experiments.
3
Measuring Cellular Metabolism with Seahorse
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The glycolytic rate and mitochondrial oxygen consumption rate were measured with the Agilent Seahorse XF Glycolytic Rate Assay kit and Agilent Seahorse XF Cell Mito Stress Test kit with an XF24 Analyzer (Seahorse Bioscience, North Billerica, MA) according the manufacturer’s guidelines. The data were normalized to the number of cells present in each well.
4
Glycolytic Rate Assay using Seahorse XFe24
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XFe24 Extracellular Flux Analyzer (Agilent, Santa Clara, CA, USA) was used to determine glycolysis metabolism. For that, cells were plated in 24-well (1.5 × 104 cells/well) Seahorse Assay plates, grown in DMEM containing 10% fetal bovine serum, L-glutamine, antibiotics, and insulin (10 µg/mL) and incubated overnight at 37 °C in a 5% CO2 humidified, 95% air incubator. The sensor cartridge was hydrated by adding 500 µL of XF Calibrant Solution (Agilent) at 37 °C in a CO2-free incubator overnight. GlycoPER was determined using the Agilent Seahorse XF Glycolytic Rate Assay Kit (Agilent, Cat No: 1033344). Data were analyzed by the software Seahorse XFe (Agilent). All measurements were normalized with the total protein concentration on each well. Each assay was run with 6 replicates per each condition.
5
Glycolytic Profiling of Pancreatic Cancer Cells
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AsPC-1 and PANC-1 cells were seeded in the XFe24 microplates and transfected with MSA or MSA/miR-1291 (5 nM for AsPC-1 cells and 20 nM for PANC-1 cells). Forty-eight hours later, cell glycolytic profile was evaluated using an Agilent Seahorse XF Glycolytic Rate Assay Kit (Agilent, Santa Clara, CA) by measuring the real-time oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) on a Seahorse XFe24 Flux Analyzer (Agilent) according to the manufacturer’s instructions. Briefly, cells were incubated with XF medium in the absence of metabolic inhibitors to record the basal OCR and ECAR levels and then rotenone (ROT) (0.5 μM) plus antimycin A (AA) (0.5 μM) and 2-deoxy-D-glucose (50 mM) were serially injected to determine basal glycolysis and compensatory glycolysis levels. Data were normalized to protein concentrations of corresponding cell lysates prepared with 0.2% SDS.
6
Glycolytic Rate Assay in PC Cells
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The XF96 Extracellular Flux Analyzer (Seahorse Bioscience, USA) was used to detect the extracellular acidification rate (ECAR) and the proton efflux rate (PER) in PC cells. Briefly, cell suspensions of PANC-1 and MIA PaCa-2 cells were seeded into the XF96 cell culture microplate (Seahorse Bioscience, USA) at a density of 3 × 104 cells prior to the experiment. Cells were incubated with base assay medium. The glycolytic rate assay was performed using the Agilent Seahorse XF Glycolytic Rate Assay Kit (103344–100; Agilent Technologies). Finally, a BCA kit (Beyotime) was adopted to calculate the concentration of protein for normalization of the results at the end of the experiments.
7
Measuring Cellular Glycolysis with Seahorse
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XFe24 Extracellular Flux Analyzer (Agilent, Santa Clara, CA, USA) was used to determine glycolysis metabolism. Cells were plated in 24-well (1.5 × 104 cells/well) Seahorse Assay plates, grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, L-glutamine, antibiotics and insulin (10 µg/mL) and incubated overnight at 37 °C in a 5% CO2 humidified, 95% air incubator. The sensor cartridge was hydrated adding 500 µL of XF Calibrant Solution (Agilent) at 37 °C in a CO2-free incubator overnight. GlycoPER was determined using the Agilent Seahorse XF Glycolytic Rate Assay Kit (Agilent, Cat No: 1033344). Data were analyzed by software Seahorse XFe (Agilent). All measurements were normalized with total protein concentration on each well. Each assay was run with 6 replicates per each condition.
8
Metabolic Profiling in Fibrosis
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Glycolytic flux and mitochondrial respiration were measured in the Seahorse XFe96 Analyzer (Agilent) using the Agilent Seahorse XF Glycolytic Rate Assay Kit in the presence of 1 lM rotenone/antimycin A and 100 mM 2DG, or the Agilent Seahorse XF Mito Stress Test Kit in the presence of 1.75 lM oligomycin, 2 lM FCCP, and 0.5 lM rotenone/antimycin A. All results were normalized to protein content.
In vivo studies Detailed methods including diet composition and duration of feeding, age, sex, and strain of animals as well as dosing information can be found in the supplemental materials. All animal procedures were in compliance with the U.S. Department of Agriculture's Animal Welfare Act (9 CFR Parts 1, 2, and 3), the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, The National Academies Press, Washington, D.C.), and the National Institutes of Health, Office of Laboratory Animal Welfare. Hydroxyproline was measured biochemically and normalized to liver weight using frozen livers that were dehydrated. FFPE slides were stained with picrosirius red or a-SMA (Abcam, ab5694 (mouse) and Biocare Medical CM001, clone 1A4 (rat)) with quantification using the Visiopharm platform. TIMP1 was measured in plasma by ELISA (R&D Systems).
In vivo studies Detailed methods including diet composition and duration of feeding, age, sex, and strain of animals as well as dosing information can be found in the supplemental materials. All animal procedures were in compliance with the U.S. Department of Agriculture's Animal Welfare Act (9 CFR Parts 1, 2, and 3), the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, The National Academies Press, Washington, D.C.), and the National Institutes of Health, Office of Laboratory Animal Welfare. Hydroxyproline was measured biochemically and normalized to liver weight using frozen livers that were dehydrated. FFPE slides were stained with picrosirius red or a-SMA (Abcam, ab5694 (mouse) and Biocare Medical CM001, clone 1A4 (rat)) with quantification using the Visiopharm platform. TIMP1 was measured in plasma by ELISA (R&D Systems).
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