Figure 1. Development of MitoRAISE protocol
A) Overall workflow of the mitochondrial ATP synthesis capacity
protocol. Samples, ranging from cell lines to patient PBMCs were
harvested in MAS buffer and permeabilized. After the cells were seeded
into a white 96-well plate and treated with the appropriate Ap5A, ADP,
and luminescence, the plate was subjected to reading. B) ATP synthesis
capacity in isolated mitochondria. Left) Dot plot showing the real time
accumulation of ATP signals from 4 µg mitochondria (circle) and MAS
buffer with 0 µg mitochondria (triangle) activated by GM and S. Arrows
indicate the time in which activating or inhibiting substrates were
injected. Right) Bar graph depicting the slope of ATP synthesis capacity
after injection of activating substrates, GM and S, to 0 µg, 2 µg, and 4
µg mitochondria. C) Bar graph depicting the slope of ATP synthesis
capacity in samples treated with 0 nM, 2 nM, 5 nM, and 10nM PMP in
10,000 cells. D) Bar graph depicting the slope of ATP synthesis capacity
in samples with <10%, 25%, 40%, and 100% of the 10,000
cells permeabilized.
GM: glutamic acid and malic acid, S: succinate, PMP: plasma membrane
permeabilizer