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.