Cell Cycle Analysis by Flow Cytometry

Flow cytometry was used to assess the results of the cell cycle analyses using propidium iodide (PI),. Following a time of 48-h of treatment using DMSO (1%) as a vehicle control and the IC₅₀ value of the D. viscosa extract. Cells were further collected by scraping and centrifugation, resuspending the cell pellet in versene buffer. Then, the fixation process with 1.8 mL 70% ethanol was carried out at 4°C for 1 h. After washing with versene buffer, the alcohol was eliminated. The final pellet was resuspended in 300 µL of PBS with 0.25 mg/mL RNAse (Type I-A, Sigma-Aldrich, Germany) and 0.1 mg/mL PI, and incubated at room temperature for 30 min in the dark. The PI fluorescence of 10,000 events was read using a FACS Canto II flow cytometer (BD Biosciences, United States). The software FlowJo 7.6.2 (Ashland, OR, United States) was used to analyze the data (Herrera et al., 2021 (link)).