4.5. Cell Cycle Analysis

MDA-MB-468 cells were seeded onto 6-well plates at a density of 3 × 10⁵ cells/mL and left overnight to attach. Culture medium was removed and the cells were exposed to 2.0 μM lutein in fresh culture medium with 10% FBS or cultured in medium with DMSO as a control. After 48 h, treated cells were stained with propidium iodide (PI) using PI/RNase Staining Buffer (BD Biosciences, BD Pharmingen, San Jose, CA, USA), according to the manufacturer’s instructions. Briefly, cells were harvested and centrifuged at 300× g for 5 min at RT, and the pellets were fixed in ice-cold 80% ethanol overnight at −20 °C. Following fixation, cells were centrifuged at 300× g for 5 min at 4 °C, washed twice in PBS, and then incubated with 0.5 ml PI/RNase solution per 1 × 10⁶ cells for 15 min in darkness at RT. Next, the stained cells were analyzed by flow cytometry using a Accuri C-6 flow cytometer (BD Biosciences) equipped with a 488-nm argon-ion laser, in order to assess the percentage of cells in phases G0/G1, S, and G2/M, based on the amount of PI incorporated into DNA; histograms of DNA distribution were then generated. The PI fluorescence intensity of individual nuclei was determined, and at least 10,000 events were measured within an acquisition rate >60 events/second. Cell cycle analyses were performed with FlowJo software (NIH). All experiments were performed in triplicate and yielded similar results.