2.4. Rhodamine 6G efflux assay

Fungal strains were cultivated in YEPD liquid medium at 200 rpm (30 °C) for 16 hours. The harvested cells were washed twice with ice-cold glucose-free PBS. Cells were suspended in ice-cold glucose-free phosphate buffered saline (PBS) and incubated at 200 rpm (30 °C) for 4 hours under starvation conditions to reduce ABC pump activity. Cells were then washed twice and diluted to 10⁸ cells/ml in ice-cold glucose-free PBS, as determined with a hemocytometer. BEA at a concentration of 16 µg/ml (about 2 × MIC) was added when necessary, while PBS was added as the negative control. All samples were incubated for another 2 hours at 200 rpm (30 °C). Then, 10 µM (final concentration) rhodamine 6G was added, and cells were incubated for further 1.5 hours at 200 rpm (30 °C). The external rhodamine 6G was then removed by washing with glucose-free PBS and glucose was added to the samples (to a final concentration of 3 mM) to reactivate the ABC efflux pumps, with PBS as the negative control. Cell samples (1 ml) were taken at designated time points, centrifuged and 100 µl of each supernatant was transferred into black 96-well microtiter plate with clear bottoms (Greiner, Germany). Rhodamine 6G fluorescence was measured with a Multilabel Plate Reader (Perkin Elmer, USA) at 510 nm excitation/535 nm emission wavelengths. Experiments were carried out at least in triplicate.