**3.4. Detection of autophagy/mitophagy using Cyto-ID® staining**

Staining of autophagic vacuoles was performed using the CytoID® autophagy detection kit (Enzolifesciences.com, Germany). GBM cells were seeded into flat-bottom 96-well microtiter plates (Sarstedt.com). One day after seeding, semi-confluent GBM cell layers were treated for 4 h with 0.01% DMSO (vehicle control) or 7 μM Vac to induce autophagy. After treatment, the culture medium was replaced by fresh culture medium containing CytoID® green detection reagent in a 10−3 dilution. After 15 min of incubation at 37°C, cells were washed twice in prewarmed Hank's balanced salt solution (HBSS). Imaging was performed by IncuCyteZOOM®. Green object count reflecting autophagosomes/autophagolysosomes was quantified and collected by IncuCyteZOOM® software.
