2.3. Induction of Autophagy

Cells that reached approximately 60% confluence were washed twice with PBS and incubated in EBSS medium (140 mM NaCl; 1 mM CaCl₂; 1 mM MgCl₂; 5 mM glucose; 20 mM HEPES (pH 7.4); 1% BSA—sterile filtrated using a 0.2 µm filter) to induce autophagy, as described previously [35]. Cells were further cultivated at 37 °C and 5% (v/v) CO₂ for various periods and then harvested for further analysis.