PI/RNase Staining Buffer

Protocol tips

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	- Cells were fixed in 70% ethanol overnight. - To stain cells were resuspended in 500 µl of PI/RNASE staining solution.

Protocol tips
- Cells were fixed in 70% ethanol overnight. - To stain cells were resuspended in 500 µl of PI/RNASE staining solution.

Publication protocol

The effect of CDK1 and CDK2 knockdown on cell cycle progression was determined by propidium iodide staining. In brief, 2 × 105 HACAT cells were synchronized in G1 by serum starvation for 48 hours. DNA content for each time point after G1 release in serum supplemented medium was determined by FACS analysis. Knockdown experiments were performed using CDK1 siRNA, CDK2 siRNA and Non-target siRNA (75 nM) according to the manufacturer's instructions. Cells were washed in PBS, fixed in 70% ethanol overnight, resuspended in 500 µl of PI/RNASE staining solution (BD Biosciences; http://www.bdbiosciences.com/eu/applications/research/apoptosis/buffers-and-ancillary-products/pirnase-staining-buffer/p/550825) and incubated for 15 min at room temperature in the dark. Samples were analyzed by FACSCalibur flow cytometry (Becton Dickinson); 10 000 events were analyzed with CellQuest software (BD Biosciences).

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