Seahorse xf cell mitochondrial stress test

Seahorse XF Cell Mitochondrial Stress Test (Agilent) was carried out as recommended based on the manufacturer’s protocol. To prepare cell samples for analysis, 10⁵ cells per well were seeded for overnight incubation in XF96 tissue culture plates supplemented with 5% CO₂. Cells were then washed twice with 200 µL of XF base medium (pre-warmed) supplemented with 2 mM glutamine, 25 mM glucose and 1 mM sodium pyruvate pH 7.4, followed by 1 h incubation in 150 µl of XF base medium plus 25 µl growth medium (175 µl final) at 37 °C without CO₂ prior to analysis. To perform the Seahorse XF Cell Mitochondrial Stress Test, the following pre-warmed reagents in the amount of 25 µl each were loaded into injector ports of the sensor cartridge calibrated on the XF96 analyzer (Seahorse Bioscience, Billerica, MA, USA): oligomycin to port A (0.8 µM final), FCCP to port B (2.5 µM), and antimycin A to port C (1 µM final). OCR was first measured under basal conditions, and additional measurements were taken after the sequential addition of oligomycin, FCCP, and antimycin A. The measurements were normalized to cell numbers based on MTT cell proliferation assays.