4.4. Caspase-3 and -7 Activity Assay

Intracellular caspase-3 and-7 activity was quantified by fluorescence microscopy using the CellEvent™Caspase-3/7 Green Detection Reagent (Life Technologies, Waltham, MA, USA). Cells were seeded in 96-well plates (Corning) after 3, 6, 12 and 24 h of cal14.1a (27 μM) and C+ (staurosporine, 1 μM) treatment. Plates were treated with 5 μM of CellEvent™Caspase-3/7 Green Detection Reagent (Life Technologies) this kit relies on the cleavage of the tetrapeptide substrate DEVD conjugated to a quenched fluorophore. 10 μg/mL of Hoechst 33342 (Life technologies) and 50 μg/mL of propidium iodide (PI) (Sigma-Aldrich) were used as reference for nucleus staining. The plates were incubated for 30 m at 37 °C with 5% CO₂ and exposed to corresponding filter (blue channel: 390-40/446-33 nm, green channel: 482-18/532-59 nm and red channel: 586-15/646-68 nm) on the fluorescence microscope.