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