Determination of apoptosis

To evaluate apoptosis, the fraction of apoptotic nuclei was quantified by fluorescence microscopy after DNA staining with Hoechst 33342. The dye was used at a final concentration of 1 µg/ml. Then, at least 100 cells per field were counted at the epifluorescence microscope (Nikon Eclipse TS100 equipped with a LED illumination system CoolLED pE-300 white and a digital camera DS-Qi1Mc) in at least three randomly selected microscopic fields. Hoechst 33342 was excited using a single-band filter set, optimized for DAPI and other like fluorophore (Semrock). The images were analysed with the macro “Apoptosis and Cell Density Macro” available in the open source image processing package Fiji/ImageJ (https://imagejdocu.tudor.lu/plugin/morphology/apoptosis_and_cell_count_macro/start) and percentage of apoptotic cells was computed.

Alternatively, induction of apoptosis was determined by analysing phosphatidylserine externalization via annexin V-FITC/PI assay (ThermoFisher Scientific). Briefly, stimulated cells were harvested (4 × 10⁵ cells/ml), washed once with PBS, and centrifuged at 300 × g for 5 min; then, the supernatant was discarded, and the pellet was resuspended in 200 µl binding buffer containing 5 µl of annexin V-FITC. Cells were incubated for 10 min at room temperature, washed once with binding buffer, and finally resuspended in 200 µl binding buffer containing 10 µL of PI. Stained cells were immediately analysed by a MACSQuant X flow cytometer (Miltenyi Biotech). Annexin V-FITC was excited with 488 nm laser and its fluorescence was acquired through 525/50 nm BP filter, while PI was excited with 488 nm laser and its fluorescence was acquired through 585/40 nm BP filter. Ten thousand events/cells were analysed per condition. Each sample was tested six times in independent experiments. The sum of early apoptosis and late apoptosis was calculated to obtain the total percentage of apoptotic cells^61,62.