Lsm 800 inverted laser scanning confocal microscope

Cells were seeded onto tissue culture plates with a glass slide and allowed to attach for 24 h. After the incubation period, the cells were washed in PBS, fixed for 15 min at RT with 4% paraformaldehyde (Sigma-Aldrich) in PBS, and permeabilized for 15 min in 0.25% Triton-X100 (Sigma-Aldrich) in PBS. The cells were then washed twice with PBS, blocked with 3% bovine serum albumin (BSA, Sigma-Aldrich) in PBS for 1 h at RT, and incubated for 1 h at 37 °C in a humidified chamber with the following primary antibodies diluted in 3% BSA in PBS-T (PBS with 0.1% (v/v) Triton X-100): rabbit anti-Aurora B antibody (Abcam, ab2254) 1:500 and mouse anti-Tubulin 1:200 (Sigma-Aldrich, T8328). The slides were washed three times with PBS-T and incubated for 1 h at 37 °C in a humidified chamber with the following secondary antibodies diluted in 3% BSA in PBS-T: goat anti-mouse IgG antibody conjugated with Fluorochrome DyLight-488 (1:250, Thermo Fisher) and goat anti-rabbit IgG antibody conjugated to Alexa Fluor 594 (1:200, Jackson ImmunoResearch). The slides were then washed twice in PBS-T and stained with 0.25 µg/ml DAPI. Images were acquired with an LSM 800 inverted laser scanning confocal microscope (Carl Zeiss) with an Airyscan detector using a × 63 1.4 NA Plan Apochromat objective (Carl Zeiss).

Candida albicans ATCC 10,231 cells were cultured in 24-well plates with a glass bottom and incubated with either DMSO or the investigated compounds in RPMI-1640 + 10% (by volume) fetal bovine serum or Spider medium (1% m/V nutrient broth (Difco), 1% m/V D-mannitol (Sigma-Aldrich, St. Louis, MO, USA), 0.2% m/V K₂HPO₄, pH 7.2) at 37 °C for 3 h. Following the incubation period, the cells were washed three times with PBS and imaged using a light microscope. Specifically, an LSM 800 inverted laser-scanning confocal microscope from Carl Zeiss (ZEISS, Göttingen, Germany), equipped with a × 63 1.4-NA Plan Apochromat objective from Carl Zeiss, was utilized for imaging.