FxCycle™ PI/RNase Staining Solution

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	- After 24h of cell seeding, cells were incubated with compounds 2–5 (25 μM) for different time periods in 6, 12, 24 or 96. - Cells were washed with 70% ethanol and placed for 24 h at −20 °C

Protocol tips
- After 24h of cell seeding, cells were incubated with compounds 2–5 (25 μM) for different time periods in 6, 12, 24 or 96. - Cells were washed with 70% ethanol and placed for 24 h at −20 °C

Publication protocol

Cells were cultured at a density of 105–106 cells per ml depending on the cell line, in accordance with ATCC's recommendations. After 24 h, cells were incubated with compounds 2–5 (25 μM) for different time periods in 6, 12, 24 or 96 well plates depending on the cell line phenotype. Then cells were washed with PBS, re-suspended in 70% ethanol and placed for 24 h at −20 °C. After centrifugation (5 min, 400g), cell pellets were washed twice with PBS then re-suspended in 500 μl of PBS, incubated in FxCycle PI/RNase Staining Solution (Life technologies, Thermo Fisher Scientific Inc. USA) and kept in the dark at room temperature for 30 min. Cellular DNA content was then assessed by capillary cytometry on a Guava EasyCyte Plus HP cytometer (EMD Millipore Corporation, MA, USA). A minimum of 10[thin space (1/6-em)]000 cells per sample was acquired and analyzed on the InCyte software. The percentage of cells in G0/G1, S, G2/M and sub-G1 was determined from DNA content histograms.

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