The Seahorse XFe/XF Analyzer was preheated on the previous day before each assay. Cells were plated in a Seahorse XF96 plate at a density of 15,000 cells per well, were placed at room temperature for 1 hour, and then incubated at 37°C and 5% CO 2 for 24 hours. In a 37°C non-CO 2 incubator, the sensor cartridge was hydrated overnight in Seahorse XF Calibrant. On the day of assay, the compounds that included glucose, oligomycin, and 2-Deoxy-D-glucose (2-DG) were loaded into appropriate ports of a hydrated sensor cartridge. Then the growth medium of cell culture was replaced with warmed assay medium using a multichannel pipette and placed in a non-CO 2 incubator at 37°C for 1 h before measured. Finally, the cells' glycolysis stress was tested using the Seahorse XFe/XF Analyzer.
Xfe xf analyzer
Manufactured by Agilent Technologies
The XFe/XF Analyzer is a laboratory instrument designed for metabolic analysis. It provides real-time measurements of cellular oxygen consumption and extracellular acidification rates to assess cellular bioenergetics.
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4 protocols using xfe xf analyzer
1
Glycolysis Stress Assay with Seahorse
2
Glycolysis Stress Measurement in Cells
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Cells were seeded into Seahorse XF96 plates and treated with glucose, oligomycin, and 2-deoxy-d-glucose (2-DG). Cells were then loaded into a hydrated sensor cartridge at appropriate ports and glycolysis stress measured on a Seahorse XFe/XF Analyzer.
3
Glycolytic Profiling of Cells
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20000 cells were incubated in Seahorse XF96 microplates with 4 µM KA or same volume of DMSO as control for 24 h and the Seahorse XFe/XF Analyzer were turned on for warming up to stabilize. Hydrate a sensor cartridge in Seahorse XF Calibrant at 37 °C in a non-CO2 incubator overnight. Cell washed twice with base medium (Seahorse bioscience) containing 2 mM glutamine, and incubated for 1 h without CO2, following three baseline ECAR (extracellular acidification rate) measurements, then 10 mM glucose (activating glycolysis), 0.5 mM oligomycin (suppressing mitochondrial ATP production and shifts the energy production to glycolysis), 50 mM 2-deoxy-glucose (2-DG, inhibiting glycolysis) were injected into cells in sequence. The XF instrument directly measures the acidification rate and reports this as ECAR, which is the standard assay for measuring glycolytic function in cells.
4
Glycolysis Stress Test in Seahorse
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Cells were plated in a Seahorse XF96 plate at a density of 15,000 cells per well, and the compounds that included glucose, oligomycin, and 2-deoxy-d -glucose (2-DG) were loaded into appropriate ports of a hydrated sensor cartridge. Finally, the cells’ glycolysis stress was tested using the Seahorse XFe/XF Analyzer.
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