Apoptosis Assay (Caspase-3 Activation)

20,000 HCT 116 cells were seeded in a black poly-D-lysine-coated 96-well plate and incubated for 24 h at 37°C. The culture medium was replaced by 100 µl DMEM-medium without phenol red (Gibco, 21063-029) supplemented with 10% FBS and 1% penicillin/streptomycin. Lichen extracts or control (DMSO) were added to the cells and incubated for 24 h at 37°C. One microliter of CellEvent Caspase-3/7 Green Detection Reagent (1:10 diluted in DMEM-medium without phenol red) was added and incubated for 90 min at 37°C (without CO₂). Afterwards, 1 µl of DRAQ5 (1:25 diluted in DMEM-medium without phenol red) was added and the cells were incubated for 30 min at room temperature. An image was taken using the ImageXpress Micro Confocal High Content Imaging System (Molecular Device, San Jose, USA). The fluorescence signal of cell nuclei was detected in the Cy5 channel (red), the signal of apoptotic cells in the fluorescein isothiocyanate (FITC) channel (green). The percentage of dead cells was determined using the “live/dead” analysis tool from Molecular Device by calculating the ratio of apoptotic cells to all cells.