2.12. Cell Cycle Analysis by Flow Cytometry

The cell cycle of HL-60 cells was analyzed using the flow cytometry assay by seeding 5 × 10⁵ cells in 6-well plates, the final volume of each well-being 1.5 mL. They were incubated at 37 °C in a humid atmosphere with 5% CO₂ until sub-confluence. After this, the cells were harvested, centrifuged and cultured again in said plate to be treated with different concentrations of PcSPs, previously dissolved in a fresh and complete culture medium. To carry out the assay, the criterion was to use three concentrations of PcSPs (0.1 mg mL⁻¹, 1 mg mL⁻¹ and 10 mg mL⁻¹), i.e., a concentration 10 times higher than the IC₅₀, the concentration IC₅₀ and another 10 times below IC₅₀. Thus, it was verified how these concentrations of PcSPs affected the HL-60 cell cycle. For the positive control, 2-methoxyestradiol (20 µM) (Sigma-Aldrich, M6383) was used. The plates were incubated under the indicated conditions for 16 h. Cells were harvested and centrifuged. The pellets were washed with PBS and fixed (70% EtOH, 1 h at −20 °C). Finally, the cells were centrifuged and washed twice with PBS, suspended in 40 μg mL⁻¹ propidium iodide staining solution (Sigma-Aldrich, P4864) and 0.1 mg mL⁻¹ RNase-A (Sigma-Aldrich, R6513) in PBS and incubated for 30 min at 37 °C protected from light. The samples were measured in the FACS VERSETM flow cytometer (BD Biosciences, San Jose, CA, USA), the results were analyzed with the BD FAC-Suite program.