2.6. Flow cytometer analysis

For quantification of DNA hypoploidy, cells were harvested by trypsinization, and ice cold 95% ethanol with 0.5% Tween 20 was added to the cell suspensions to a final concentration of 70% ethanol. Fixed cells were pelleted, and washed in 1% bovine serum albumin (BSA)‐PBS solution. Cells were resuspended in 1 mL PBS containing 20 μg/mL RNase A, incubated at 4°C for 30 min, washed once with BSA‐PBS, and resuspended in PI solution (10 μg/mL). After cells were incubated at 4°C for 5 min in the dark, DNA content were measured on a CYTOMICS FC500 flow cytometry system (Beckman Coulter, FL, California) and data was analyzed using the Multicycle software which allowed a simultaneous estimation of cell‐cycle parameters and apoptosis. To quantify the development of acidic vesicular organelles (AVOs), the cells were stained with acridine orange (1 μg/mL) for 15 min, removed from the plate with trypsin‐EDTA, and analyzed using a FACScan flow cytometer. For autophagy inhibition, cells were pretreated with 1 mM 3‐MA for 1 h and incubated with Embelin for 24 h.