Multicycle software for dna content and cell cycle analysis

Nuclear DNA fragmentation was quantified by flow cytometry of hypodiploic (subG1) DNA after cell fixation and PI staining [30 (link), 31 (link)]. Briefly, cells were washed with PBS, pelleted and fixed in ice cold ethanol/water (70/30, v/v) for 1 h, pelleted again and washed twice with PBS, and finally resuspended in PBS containing RNAse (20 μg/mL) and PI (100 μg/mL). Events in the different cell cycle phases were gated manually using an EPICS XL cytofluorimeter (Beckman Coulter, Hialeah, Fl, USA). At least 10.000 events/sample were acquired. Collected data were analyzed using the Multicycle software for DNA content and cell cycle analysis (Phoenix Flow System, San Diego, CA, USA). The subG1 events representative of the apoptotic cells, and the events in the other cell cycle phases, are given as percentage of the total cell population.
Membrane permeability, indicative of cell death, was investigated by resuspending the cells in HBSS containing PI (200 μg/mL) at room temperature and analyzed by flow cytometry (EPICS-XL), measuring the fluorescence emission at >575 nm (FL3).

Nuclear DNA fragmentation was quantified by flow cytometry evaluation of hypodiploic (subG1) DNA events after cell fixation and PI staining.^{27 (link),40 (link)} Briefly, cells were washed with PBS, pelletted and fixed in ice cold ethanol/water (70/30, v/v) for 1 h, pelletted again, washed twice with PBS, and finally resuspended in PBS containing RNAse (20 μg/ml) and PI (100 μg/ml). Events in the different cell cycle phases were gated manually using an EPICS XL cytofluorimeter (Beckman Coulter, Hialeah, Fl, USA). At least 10.000 events/sample were acquired. Collected data were analyzed using the Multicycle software for DNA content and cell cycle analysis (Phoenix Flow System, San Diego, CA, USA). SubG1 events, representative of apoptotic cells, are given as percentage of the total cell population.
Membrane permeability, indicative of cell death, was investigated by resuspending the cells in HBSS containing PI (20 μg/ml) at room temperature and analyzed by flow cytometry (EPICS-XL), measuring the fluorescence emission at >575 nm (FL3log).