Cell cycle analysis by flow cytometry using propidium iodide

After trypsinization, the cells were centrifuged, resuspended in 70% ethanol and fixed at 4°C for ≥24 h. To detect apoptotic cells that could be suspended in the culture medium, the media from all plates were collected and centrifuged. The resulting pellets were processed together with the rest of the cells on the plates. For propidium iodide labeling, the cells were resuspended and stained with a solution of 3.4 mM sodium citrate (PanReac Quimica SLU), 100 µg/ml RNAse A (Roche Diagnostics GmbH) and 20 µg/ml propidium iodide (Roche Diagnostics GmbH), and incubated in the dark at room temperature for 1 h.

For data acquisition, a total of 20,000 events were counted using FACScan Mod and FACScalibur flow cytometer (BD Biosciences). The obtained data were analyzed using CellQuest 3.2 software (BD Immunocytometry Systems).