Las software v4

MBVs’ effect on NHDF cell morphology was evaluated using phalloidin-DAPI immunofluorescence staining. Cells were seeded in 24-well plates over microscope glass slides, at a concentration of 14000 cells/well. After 24 h, the culture medium was replaced with MBV-containing medium; specifically, MBVs previously diluted in PBS at different concentrations of 9 × 10⁷ MBVs/mL, 3.6 × 10⁷ MBVs/mL, and 1.8 × 10⁷ MBVs/mL. A total of 750 μL of PBS containing MBVs was placed in each well, and 750 μL of culture medium was added. In order to obtain a representative control, a medium/PBS in a ratio of 1:1 was used. At time points of 1 and 3 days after MBVs were added to cells, samples were fixed using 4% formaldehyde for 1 h at room temperature. Phalloidin, Fluorescein Isothiocyanate Labeled (Sigma-Aldrich, P5282, Milan, Italy) was placed over the samples and incubated at 37 °C for 45 min. Nuclei were stained with 1 μg/mL DAPI (Sigma-Aldrich, Milan, Italy) for 1 min. Stained cells were observed using a fluorescence microscope (Leica DM700, Wetzlar, Germany), and images were acquired via Leica software LAS V4.7 (Leica, Wetzlar, Germany). Experiments were performed in triplicate.