3.2 Development of MitoRAISE using isolated mitochondria and
viable cells
We first performed the MitoRAISE on isolated mitochondria of A549 cells
(Figure 1B). The concentration of the mitochondria was measured through
BCA protein quantification. As expected, the ATP synthesis capacity of
isolated mitochondria showed correlation with the concentration of the
isolated mitochondria. With the doubling of concentration of isolated
mitochondria from 2 μg to 4 μg, the ATP synthesis capacity induced by
the activators, glutamic acid and malic acid (GM) and succinate (S),
also doubled. The addition of GM or S to the mitochondrial assay
solution (MAS) buffer with 0 μg mitochondria showed no reaction. We next
sought to measure the mitochondrial ATP synthesis capacity from viable
cells. To overcome the challenge of inducing the entry of substrates
into the cells, we utilized the plasma membrane permeabilizer (PMP). PMP
can efficiently induce cellular membrane permeabilization while causing
limited mitochondrial damage. 5 nM of PMP was sufficient to fully
(100%) permeabilize 1*10^4 cells (Figure 1C, Supporting Figure 1A).
To test the importance of permeabilization on finding the full ATP
synthesis capacity of cells, we intentionally mixed permeabilized cells
with intact cells to obtain <10%, 25%, 40%, and 100%
permeabilized populations (Supporting Figure 1B). There was a
permeabilization percentage dependent increase in ATP synthesis capacity
(Figure 1D). This means that cellular permeability for the substrate to
enter the cells is important for ATP measurement.