Agilent seahorse xf cell energy phenotype test kit

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.

One day prior to the actual experiment, 200 μL/well of distilled water was dispersed in the utility plate. The cartridge sensors (Seahorse mini Fluxpak XFe96, 102601–100, Agilent Technologies) were hydrated, overnight, in a 37°C non-CO₂ incubator. On the day of the experiment, distilled water was replaced by XF Calibrant (100840–100, Agilent Technologies); cartridge sensors were immersed into the XF Calibrant and incubated for 1 hour in a 37°C non-CO₂ incubator. A total of 180 μL of XF Calibrant was added in the 4 edge wells in the culture plate. The cover guide was then loaded on the CD8⁺ T cell–seeded culture plate. A total of 20 μL of oligomycin (port A) and 22 μL of FCCP (port B) (from Agilent Seahorse XF Cell Energy Phenotype Test Kit, 103325–100, Agilent Technologies) were then displayed, at a final concentration of 1 μM and 1.5 μM, respectively. The utility plate filled with XF Calibrant and capped with the cartridge was positioned in the Seahorse XFe96 analyzer’s tray (Agilent Technologies), and the calibration of the signals generated by all 96 wells was performed. The culture plate was then introduced in the tray and the acquisition program was started. Data were analyzed through the Wave 2.6.1 Software (Agilent Technologies).

The Mitochondrial Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in MDA-MB-231 cells were assessed by Agilent Seahorse XF Cell Mito Stress Test Kit and Agilent Seahorse XF Cell Energy Phenotype Test Kit (Agilent Technologies) and run in Agilent Seahorse XFe96 Analyzer (Seahorse Bioscience) as per manufacturers protocol. Cells were seeded at density (5 × 10³) cells/well in Seahorse XF Cell Culture Microplate, transfected after 24 h and assay was completed 48 h after transfection.