Mitochondrial Activity Assessment in Yeast

The mitochondrial activity was monitored using the Rhodamine 123 (Sigma-Aldrich) and Mitotracker Green FM (Sigma-Aldrich) fluorescent dyes. Yeast cells were stained according to the manufacturer’s instructions. Initially, yeast cells were harvested by centrifugation of 8000 × g for 10 min at 4°C, diluted in PBS 1X to 10⁶ cells/mL and stained during 45 min at 37°C with Mitotracker (400nM). Then, the cells were washed with PBS 1 X and labeled with Rhodamine (2.4 μM) for 45 min at 37°C. Afterward, the cells were washed three times with PBS 1X and analyzed by fluorescence microscopy (Zeiss Axiocam MRc – Scope A1) at 450–490 nm for Mitotracker (FS09) and 515–575 nm filter (FS15) for Rhodamine. All experiments were performed in triplicate and the minimum of 50 cells for each microscope slides were assessed to measure of the fluorescence intensity (in pixels). Student’s t-test and p ≤ 0.05 were considered statistically significant as described by Chaves et al. (2019) (link).