2.2 Mitochondrial ATP contents and Response Against Inhibiting
and Stimulating Substrates (MitoRAISE)
Cells or PBMCs were seeded into a 96-well white plate along with ATP
standard material in 1/2 serial dilutions to construct a standard curve.
Up to 50 µL mitochondrial assay solution (MAS) buffer containing 250 μM
Ap5A was injected and placed on a plate shaker for 2 min. Next, 50 µL of
ATPlite solution with 100 μM ADP was added and the mixture was again
placed on a plate shaker for 2 min. After incubation for 10 min,
luminescence signals were read five times at 2-minute intervals; 10 µL
of activating substrates (10 mM glutamic acid mixed with 10 mM malic
acid for complex I and 100 mM succinate for complex II) were added to
the wells, and the plate was read five times at 2-minute intervals.
Finally, 10 µL of inhibiting substrates (40 µM rotenone for complex I
and 800 µM malonate for complex II) were injected into the wells, and
the plate was read five times at 2-minute intervals. The values of the
samples were adjusted against the ATP standard curve, and we defined the
ATP synthesis capacity as the slope of increasing ATP luminescence
signal. The reduction of the slope after injection of the inhibitory
substrates was defined as the sensitivity to inhibitory substrates.