Fxcycle pi rnase staining

Samples were fixed in 70% ethanol overnight, washed with PBS, treated with FxCycle PI/RNase Staining (Life Technologies) solution for 20 min and analyzed by FACS. For phospho-histone-H3 analysis, fixed cells were washed 2X in PBS-BSA 5%-Triton 0.1%, incubated with phospho-histone-H3 antibody (Cell Signaling Technology) for 2hours. After 2X wash with PBS-BSA5%-Triton0.1%, samples were incubated with Alexa secondary antibodies (Life Technologies), for two more hr. The samples were then washed 3X in PBS before analysis.

To investigate the influence of CX-4945 on the cell cycle the progenitor cells were cultured for 5 days to generate sufficient amounts of cells. Initially, they were dissociated as described and seeded at a density of 1 million cells per 5 ml medium. Cells were treated with 10 or 20 μM CX-4945 for 24 h in proliferation medium. As controls, DMSO and the usual proliferation medium were used. After 24 h, the cells were harvested for cell cycle analysis. Cells were centrifuged at 120xg for 5 min and dissociated carefully with accutase to avoid clustering during the cell cycle analysis.
Cells were centrifuged, the supernatant was discarded and 2 ml cold 70% ethanol per 1 million cells were added with gentle vortexing. Cells were stored for 2 h at 4 °C. Subsequently, the samples were centrifuged at 300xg for 1 min to remove the ethanol. To wash the cells 1 ml PBS was added and cells centrifuged again as described before. After this washing procedure, cells were stained with FxCycle PI/RNase staining (Life technologies) solution and incubated for 15 to 30 min at room temperature.