Seahorse assays were conducted in accordance with a published protocol (50 (link)). Mitochondrial bioenergetic measurements were performed using the Seahorse XFe96 Extracellular Flow Analyzer (Agilent Technologies, Santa Clara, CA, USA) to measure the oxygen consumption rate (OCR) in different respiratory states. 2.5 x 104 LS174T cells in 80 μL medium were seeded in XF96 Cell Culture microplate and cultured overnight. Then cell culture media were replaced with Seahorse XF modified media before cells were treated. In the standard assay, cells were treated sequentially with 1 μM oligomycin, 1.0 μM FCCP, and a mixture of 1.0 μM rotenone and 1.0 μM antimycin A. To determine if compounds inhibited ETC complex V/ATP synthase, oligomycin was replaced with DMSO, or compounds dissolved in DMSO. To test whether compounds were ETC complex I or III inhibitors, rotenone and antimycin A were replaced with DMSO, or compounds in DMSO solution.
Seahorse xfe96 extracellular flow analyzer
Manufactured by Agilent Technologies
Sourced in United States
The Seahorse XFe96 Extracellular Flow Analyzer is a laboratory instrument used to measure the metabolic activity of cells. It provides real-time analysis of cellular respiration and glycolysis through the detection of changes in oxygen and pH levels in the cell culture media.
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2 protocols using seahorse xfe96 extracellular flow analyzer
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Mitochondrial Bioenergetics Profiling using Seahorse Assay
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Standardized Seahorse Assay Protocol
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The Seahorse assays were conducted following a standardized protocol [36 (link)]. The Seahorse XFe96 Extracellular Flow Analyzer (Agilent Technologies, Santa Clara, CA, USA) was used to measure the oxygen consumption. An XF96 Cell Culture microplate was used to culture the LS174T cells, with a seeding density of 2.5 × 104 cells in 80 μL medium. The plate was incubated overnight. On the next day, the cell culture media was replaced with Seahorse XF modified media. Oligomycin (1 μmol/L), FCCP (1 μmol/L), and a mixture of rotenone (1 μmol/L) and antimycin A (1 μmol/L) were sequentially added during the measurements. To identify whether the compounds were ETC complex V/ATP synthase inhibitors, an equal volume of DMSO or compounds dissolved in DMSO solution was used as a substitute for oligomycin. To assess the inhibitory effects of the compounds on ETC complex I or III, DMSO alone was used as a substitute for rotenone and antimycin A, or these compounds were dissolved in DMSO.
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