PI/RNase Staining Buffer

Protocol tips

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	- Cells were fixed in 70% cold ethanol overnight at -20°C. - After staining, cells were Incubate on ice until measured.

Protocol tips
- Cells were fixed in 70% cold ethanol overnight at -20°C. - After staining, cells were Incubate on ice until measured.

Publication protocol

Approximately 1 × 105 cells/ml were seeded in 10% FBS-DMEM in 100-mm dishes and incubated overnight. The medium was changed to FBS-free DMEM for 24 h and then changed to 1% FBS-DMEM for an additional 24 h. The cells were trypsinized and washed twice with phosphate-buffered saline (1 × PBS), autoclaved, and then fixed in 70% cold ethanol overnight at -20°C. Immediately before the assay, the cells were stained using PI/RNase staining buffer (Becton Dickinson, 550825) in the dark on ice until cell cycle analysis was carried out using a flow cytometer (Becton Dickinson, BD FACS Canto II). The flow cytometer equipped with a 488-nm laser was used for the analysis and 10,000 cells were analyzed in each measurement. Data were acquired and analyzed using the BD FACS Diva software (Becton Dickinson). The relative change in the mean fluorescence intensity was calculated as the ratio between mean fluorescence intensity in the channel of the treated cells and that of the control cells.

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