Cell cycle analysis by flow cytometry

On day 0, SH-SY5Y cells were plated in a 6-well plate at a density of 1.1×10⁵ cells/well. On day 1, 80 µl of phenformin, to a final concentration of 0.1, 0.5, 1, 2 and 5 mM, was added to the respective wells and incubated at 37°C in 5% CO₂ for 72 h. Following treatment, the cells were trypsinised and pelleted by centrifugation. The cells were subsequently fixed in 70% ethanol/PBS and incubated overnight at 4°C. The ethanol was removed using centrifugation at 300 × g for 5 min at room temperature, and the pellets were resuspended in 400 µl PBS supplemented with 10 µg/ml RNase A (Sigma Aldrich; Merck KGaA) and propidium iodide (PI) 100 µg/ml (Sigma Aldrich; Merck KGaA). The cells were then incubated in the dark for 30 min at 37°C. Cell cycle distribution was analysed using the FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) using Cell Quest Pro (version 4.3; BD Biosciences).