Seahorse XFp Assay for T-cell Metabolic Profiling

On the day prior to the assay, the Agilent Seahorse XFp Sensor Cartridge with XF Calibrant was hydrated in a non-CO2 37°C incubator overnight. Isolated T cells co-treated with eicosenoate were stimulated with ImmunoCult Human CD3/CD28 T-cell Activator (StemCell Technologies, Vancouver, Canada) for 24 h. On the day of the assay, cells were resuspended in warmed assay medium to the desired concentration (5×10⁵ cells in 50μl/well) before seeding them onto the CellTak-coated Seahorse Cell Culture Miniplate (wells A and H were used as background correction wells). Then, cells were centrifuged at 350×g for 5 mins, and 130μl assay medium was added to each well for a final volume of 180μl. Finally, the Miniplate was transferred to a non-CO2 37°C incubator for 20 mins to ensure that the cells were entirely stable. Oxygen consumption rate (OCR) was measured without adding any drug (basal respiration), followed by measurement of OCR changes upon subsequent addition of 1.5 μM ATP synthase inhibitor oligomycin and 1 μM carbonyl cyanide-4 (trifluoromethoxy), and phenylhydrazone (FCCP). Finally, 0.5 μM rotenone and 0.5 μM antimycin A were injected to completely inhibit mitochondrial respiration by blocking complex I and complex III. Basal respiration, maximal respiration, and spare respiration were analyzed using an XFp Cell Mito Stress Tests Kit on an Agilent Seahorse XF HS Mini instrument, according to the corresponding operation protocol.