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Seahorse assay

KV Ksenija Velickovic
DW Declan Wayne
HL Hilda Anaid Lugo Leija
IB Ian Bloor
DM David E. Morris
JL James Law
HB Helen Budge
HS Harold Sacks
MS Michael E. Symonds
VS Virginie Sottile
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Cells were seeded into the XFe96 Microplates (Seahorse Bioscience) to measure the oxygen consumption rate (OCR: an indicator of mitochondrial respiration) and the extracellular acidification rate (ECAR: an indicator of glycolysis) as previously described20. OCR and ECAR readings were measured under basal conditions and after the addition of mitochondrial inhibitors (oligomycin, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) and rotenone/antimycin A). Coupling efficiency, an indicator of the proportion of respiratory activity used to make ATP, was determined by calculating the percentage of OCR immediately following the oligomycin treatment with the final baseline value. Seahorse datasets were normalised to cell number using Hoechst 33258 fluorescent dye signal measured immediately after Seahorse analysis31.

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