ATP Quantification Assay

The quantification of intracellular ATP was performed using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega Corporation, USA), which is based on the reaction between the luciferase from Photuris pennsylvanica (LucPpe2) and ATP molecules generating a luminescent signal. C. albicans and C. krusei cells (10⁶ CFU) were treated for 24 h at 37°C with TesTI at MFC, MIC, aaa MIC, and bbb MIC. A 100% growth control (non-treated cells) was also performed as described in Section “Antifungal Assay.” After this period, the microplate was maintained for 30 min at 25°C and then the cells were transferred to wells of an opaque-walled 96-well plate (100 μL per well). Next, 100 μL of Cell Titer-Glo^® reagent was added to each well and the contents were mixed for 2 min on an orbital shaker to induce cell lysis. The plate was incubated for 10 min at 25°C and then the luminescence was recorded. Three independent experiments were performed in triplicate. The results were expressed as relative ATP levels, which were calculated by the ratio between the level in the test assay and that in the control assay.