8 μL of this suspension was added to 200 mL of liquid medium and incubated in a shaker (200 rpm) bath for 16 h at 30 °C. The culture was then centrifuged at 3000 x g for 5 min at 4 °C, the supernatant was discarded and 20 mL of Milli-Q water was added to the pelleted cells, which were suspended and centrifuged again. This process was repeated three times. Finally the yeast cells were resuspended
in 3 mL of Milli-Q water. Aliquots of 107 cells were pre-incubated for 30 min in 800 μL of protein or peptide samples in 10 mM Tris–HCl, Bcl-2 inhibitor pH 6.0 or the dilution buffer alone. After pre-incubation, 200 μL of 500 mM glucose was added to the cells and the medium pH was measured every minute for 30 min. The amount of H+ released by the cell was calculated as the difference between the initial pH and the final pH (ΔpH), considering the equation pH = −log [H+]. The values are averages of triplicates for each experiment. The permeabilization of the plasma membrane was assessed by measuring absorption of SYTOX Green (Invitrogen, Grand Island, NY, USA) as described by [22]. This dye forms a fluorescent complex with nucleic acids, entering cells when the integrity of their plasma membrane is compromised. Fungal
cells were incubated with different concentrations of the test samples for 24 h and then exposed to 0.2 μM SYTOX Green for 30 min at room temperature. Obeticholic Acid order The cells were observed under a microscope (Axioskop 40 – Zeiss) equipped with a filter for fluorescein detection (excitation wavelength 450–490 nm and emission 500 nm). Fluorescent probes (LIVE/DEAD® Yeast Viability Kit – Invitrogen, Grand Island, NY, USA) were used to evaluate the viability and metabolic activity of yeasts in the presence of test samples. The yeast cells were grown overnight (16 h) in Sabouraud medium at 28 °C in the presence of either JBU, Jaburetox, dialysis buffer, or H2O2, and then centrifuged (3000 × g, 10 min) to remove the medium. Yeasts were suspended in buffer GH (2%
d-(+) glucose, 10 mM Na-HEPES, pH 7.2) and then 1 μM of FUN-1 and 12.5 μM of calcofluor were added. After more 2 h of incubation at 28 °C, the cells were viewed under a fluorescence microscope (Axioskop 40 – Zeiss) equipped with filters for different wavelengths to allow visualization of fluorescein (green), rhodamine (red) and DAPI (blue). Liothyronine Sodium Alternatively, cells were grown overnight in Sabouraud medium at 28 °C in the absence of test samples, followed by centrifugation to remove the medium. Yeasts were suspended in buffer GH (2% d-(+) glucose, 10 mM Na+-HEPES, pH 7.2). 100 μL of cell suspension were mixed with the samples JBU, Jaburetox, dialysis buffer, or H2O2, and maintained for 2 h at 28 °C. Then, 1 μM of FUN-1 and 12.5 μM of calcofluor were added and after 2 h at 28 °C, the cells were viewed under the microscope Axioskop 40 – Zeiss with different filters, as described above. All experiments were run in triplicates. Data were evaluated using “one-way” ANOVA followed by the t test of Bonferroni or Dunnett.