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.