2.3. Cell-Cycle and Apoptosis Analysis

CaSki and C33A cells were seeded into 6-well plates at a density of 1 × 10⁶ cells per well and exposed to 10 mM metformin with or without 20 μM everolimus for 48 h. Cells were then collected and fixed with 70% ice-cold ethanol at −20 °C overnight. After fixation, cells were centrifuged at 400× g for 10 min at 4 °C, washed with cold PBS, and stained with 0.5 mL PI/RNases staining buffer (PI, 10 μg/mL; RNases, 300 μg/mL; #550825, BD Pharmingen; BD Biosciences, Franklin Lakes, NJ, USA) for 15 min at room temperature in the dark. Cell apoptosis was detected using an FITC Annexin V apoptosis detection kit (#556547, BD Biosciences); cells were double-stained with 5 μL FITC Annexin V (20 μg/mL) and 5 μL propidium iodide (PI, 50 μg/mL) for 15 min at RT in the dark. Finally, the stained cells were analyzed using FC500 flow cytometry and CXP software (version 2.3; Beckman Coulter, Brea, CA, USA), and early and late apoptotic or necrotic cells were assessed.