Determination of cell mortality
To determine cell viability, cells treated by 200 μM
AlCl3 were stained at 24 h with Evans Blue (Gaff &
Okong’O-Ogola, 1971). Cells were transferred into a custom-made staining
chamber to remove the medium, and then incubated with 2.5% Evans Blue
for 3-5 min. After washing three times with distilled water, cells were
mounted on a slide and observed under a light microscope (Zeiss-Axioskop
2 FS, Differential Interference Contrast, 20 × objective). Evans Blue is
membrane impermeable, but can penetrate into dead cells due to the
breakdown of the plasma membrane, resulting in a blue staining of the
cell interior. Mortality was determined as ratio of the number of dead
cells over the total number of scored cells. For each time point, 1500
cells were scored in three dependent experiments.