Propidium iodide rnase a stain

For cell-cycle analysis, 48-hour cisplatinum-treated, 48-hour oncotic decay, 36-hour colchicine-treated, or untreated control cells were fixed in 70% (v/v) ethanol (Commercial Alcohols, Brampton, ON) for 1 hour at −20°C. Cells were permeablized by 0.1% (w/v) Triton-X100 (Sigma Aldrich, St. Louis, MO) for 2 minutes at room temperature, and then incubated with propidium iodide/RNase A stain (Molecular Probes, Eugene, OR) for 30 minutes at 37°C in the dark.
For apoptosis-oncosis characterization, 72-hour cisplatinum-treated, 72-hour oncotic decay, or untreated control cells were stained with a viability marker such as propidium iodide (PI), then backgated onto a color dot plot of forward versus side scatter based on PI fluorescence. Cell debris characterized by a very low FSC/SSC and PI negative stain, and were excluded prior to backgating. Doublets were also excluded by forward scatter signal width versus area analysis.
Flow cytometry measurements were performed using a BD LSRII flow cytometer (BD Sciences, San Jose, CA), with 488 nm light exciting PI to emit at a wavelength of 610 nm, captured through the TexasRed bandpass filter. All experiments captured a minimum of 50,000 events per sample.
Cell cycle analysis was performed using FCS Express 5 Multicycle Software (De Novo Software, Glendale, CA).

For each of the treatment times or time-matched control, cells were dissociated from their flasks by trypsin and then fixed in 4% (w/v) paraformaldehyde (Fischer Scientific, Mississauga, ON) for 60 minutes at 4°C. Cells were permeablized by 0.2% (w/v) Triton-X100 (Sigma Aldrich, St. Louis, MO) for 5 minutes at room temperature, and then incubated with propidium iodide/RNase A stain (Molecular Probes, Eugene, OR) for 30 minutes at 37°C in the dark.
Flow cytometry measurements were performed using a BD LSRII flow cytometer (BD Sciences, San Jose, CA), with 488nm light exciting PI to emit at a wavelength of 610nm, captured through the TexasRed bandpass filter. Cell cycle analysis was performed using FCS Express 4 Multicycle software (De Novo Software, Glendale, CA).