To assess cellular apoptosis (programmed cell death), we used the in Situ Cell Death Detection Kit (Roche), Fluorescein. In brief, coverslips with adherent cells were fixed and permeabilized (see Immunofluorescence) following incubation in a nucleotide mixture containing fluorescein-dUTP and TdT (terminal transferase), according to the manufacturer’s instructions. Cell nuclei were detected by incubation with DAPI. Micrographs were digitally recorded with a Leica DM6000 FS fluorescence microscope. TUNEL-positive cells appeared green, whereas TUNEL-negative nuclei appeared blue. Negative controls were incubated without TdT. The percentage of TUNEL-positive cells vs. TUNEL-negative cells was quantified for each experimental condition.
