3. Results
3.1 Mitochondrial ATP contents and Response Against Inhibiting and Stimulating Substrates (MitoRAISE) assay development
MitoRAISE is a quick and efficient workflow for the direct and simultaneous measurement of ATP contents, ATP synthesis capacity, and response to inhibitory substrates in less than 2 hours. Figure 1A describes the overall workflow. ①~④ Cells are permeabilized, treated with adenylate kinase and go through basal reading. The basal luminescence represents the amount of ATP in the sample at the time of measurement. MitoRAISE can simultaneously measures the rate at which ATP is produced in real time after treatment of various activating substrates of the ATP synthesis process. ⑤~⑥ ATP synthesis capacity is expected to reflect the status of mitochondria more accurately because it represents the efficiency of the mitochondrial function. The increasing signal after the injection of activating substrates was labeled ATP synthesis capacity. After activation, inhibitory substrates are added, and the mitochondrial sensitivity to inhibitory substance is calculated as the subtraction of inhibitory slope from the activation slope. Finding the sensitivity to inhibitory substance can further illustrate the ability of the mitochondrial function as it will show how much the inhibitory substance can block mitochondrial ATP synthesis capacity. This would hint the amount of which the pathway related to the inhibitory substrate has been damaged.