Las af ver 3

Observations were made in an inverted
microscope (Model DMi8, Leica Microsystems, Germany) equipped with
fluorescence filters (546/10 RHOD excitation filter and emission 585/40,
350/50 DAPI filter and emission 460/40, and 480/40 FITC filter and
527/30 emission), a cooled monochromatic DFC 450C camera (Leica Microsystems,
Germany), and fluorescence overlay software (LAS AF ver. 3.1.0, Leica
Microsystems CMS GmbH, Germany).
Solutions of the compounds
were prepared in DMSO and then diluted 1:10 with BHI culture medium
obtaining a concentration of 200 μg/mL. This solution (150 μL)
was added to wells in a 96-well microtiter plate and 50 μL of
bacterial suspension (1 × 10⁸ CFU/mL). The plates
were incubated at 37 °C for 24 h, and the supernatant was removed
and stained with the fluorescent dyes.

Signals emitted in the Live/Dead Kit (Sigma-Aldrich 04511) and ROS Kit (Sigma-Aldrich MAK 143) assays used to examine the mechanisms of cell death and proliferation were analyzed in an inverted epifluorescence microscope (Model DMi8 , Leica Microsystems, Germany) equipped with fluorescence filters (546/10 RHOD excitation filter and 585/40 emission, DAPI 350/50 excitation filter and 460/40 emission, and FITC excitation filter 480/40 and emission 527/30), a monochrome DFC 450C camera (Leica Microsystems) and fluorescence overlay software (LAS AF ver. 3.1.0, Leica Microsystems CMS GmbH). Since the ommochromes are capable of absorbing ultraviolet rays and can interact with ultraviolet light, the fluorescence emission in the suspension of methanol/HCl pigments (0.31-10 mg/ml) in nutrient broth medium (pH 7.4) was evaluated.

Propidium iodide is a fluorescent stain for nucleic acids and cell membrane integrity and excludes PI from staining viable and apoptotic or damaged cells. In a 96-well microplate, 100 μL of yeast suspension at a concentration of 2 × 10⁶ yeast/mL in PDB was dispensed and incubated at 37 ± 2 °C for 3 h. After this incubation period, 100 μL of different nanoparticles and controls was added and incubated for 24 h under the same temperature conditions. Following the 24 h incubation, 10 μL of 3 μM propidium iodide (PI) was added, and the samples were again incubated at the same temperature for 1 h. After incubation, cells were analyzed using a fluorescence microscope (Model DMi8; Leica Microsystems, Wetzlar, Germany) equipped with fluorescence filters (546/10 excitation filter for PI and 585/40 emission filter), a cooled monochromatic DFC450 C camera (Leica Microsystems, Wetzlar, Germany), and fluorescence overlay software (LAS AF ver. 3.1.0; Leica Microsystems CMS GmbH, Mannheim, Germany).