Fluorescein diacetate/propidium iodide (FDA/PI) staining

Cells were seeded on coverslips. After overnight, cells were treated with different conditions for 3 h and then incubated in normal medium for 21 h. For staining of live and dead cells, cultures were incubated with normal medium containing FDA (3 mg/ml) and PI (5 mg/ml) for 5 min. Live (FDA positive) and dead (PI-positive) cells were observed and counted with a fluorescent microscope (Zeiss Axio Observer Z1) at excitation/emission wavelengths of 470 nm/535 nm for FDA and 585 nm/615 nm for PI.