BD Cycletest™ Plus DNA Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- After cells were seeded, they were treated with control or SP-6-27 treated (0.5 uM, 24h)	- PI staining was performed for 10 min at 4°C

Upstream tips
- After cells were seeded, they were treated with control or SP-6-27 treated (0.5 uM, 24h)
Protocol tips
- PI staining was performed for 10 min at 4°C

Publication protocol

In order to detect the distribution of cells within the different phases of cell cycle, cells were stained with PI using BD Cycletest™ Plus DNA kit (BD Biosciences, USA) and analyzed by flow cytometry. Briefly, 0.5 × 106 cells of control or SP-6-27 treated (0.5 uM, 24h) cells were trypsinized and washed with PBS. Cells were treated with a series of buffers for digestion of cell membranes, cytoskeletons and RNA according to the manufacturer instructions. PI staining was performed for 10 min at 4°C. Immediately, the fluorescence emitted from the PI–DNA was measured for individual cells using a flow cytometer (BD, FACS Calibur). The data was acquired using BD CellFIT software. Data from three experiments (in triplicate) were collected and mean ± S.D. of the percentage of cells in the G2/M phase were calculated.

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