Cell cycle analysis and mitotic arrest

For cell cycle analysis, cells were harvested, washed once in ice-cold phosphate buffered saline (PBS), and fixed overnight at –20°C with 80% ethanol in PBS. Cells were washed three times with PBS, and suspended in PBS containing 0.1% Triton X-100, 25 μg/mL propidium iodide (PI, Molecular Probes), and 0.2 mg/mL RNase A (Sigma). Samples were stained at 4°C overnight and stored at the same temperature until analysis by LSR Fortessa (BD Biosciences) flow cytometer. Results were analyzed using FlowJo (Treestar).

To arrest MDA-MB-468 cells in mitosis, cells underwent a single thymidine block (2 mM thymidine, 16–24 hours), a 3-hour release, followed by treatment with 10 μM S-trityl-L-cysteine for 10 hours. Roughly 50% of MDA-MB-468 cells would float and arrest in mitosis using this method.