2.12. Measurement of Intracellular and Extracellular ATP Levels

The ATP level was determined with a bioluminescence assay using an ATP Determination Kit (A22066, Invitrogen). Briefly, C. albicans cells were diluted to a final concentration of 3 × 10⁸ CFU/mL with 12.5 mM sodium acetate, incubated with or without different concentrations of LfcinB15 at 37 °C for 1 h, and collected by centrifugation. The supernatant was kept on ice before use for measuring the extracellular ATP level, while the cell pellets were broken, as previously described [30], for measuring intracellular ATP.

For ATP measurement, the ATP Determination Kit was used according to the manufacturer’s instructions (Invitrogen). Briefly, 1× reaction buffer containing 0.5 mM D-luciferin, 1.25 μg/mL firefly luciferase, and 1 mM DTT was freshly prepared before each experiment, and 10 µL of the supernatant or cell lysate was mixed with 90 µL of the reaction buffer. Then, the mixture was placed into each well of a black 96-well microplate, and luminescence was read using a Victor3™ Plate Reader (PerkinElmer) at 560 nm. A standard curve of increasing ATP concentrations (0, 0.01, 0.1, 1 μM) was created. Signals represented at least three independent experiments were obtained and the ATP concentration of each sample was calculated from the standard curve.