Cell Cycle Phase Determination Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cells were seeded and treated with concentrations of the EtOAc fraction for 12 h.	- Cells were fixed and incubated for more than 2h.

Upstream tips
- Cells were seeded and treated with concentrations of the EtOAc fraction for 12 h.
Protocol tips
- Cells were fixed and incubated for more than 2h.

Publication protocol

The cell cycle phase was assayed by DNA fragment staining with PI using the Cell Cycle Phase determination kit (Cayman Chemical, MI, USA), according to the manufacturer’s instructions. AGS cells (4×105 cells/ml in a 24-well plate) were added to different concentrations of the EtOAc fraction for 12 h and then harvested. After centrifugation, the pellets were washed and suspended in cell-based assay buffer. The cells were fixed and permeabilized by adding 1 ml of a fixative to each tube for more than 2 h. After centrifugation, the fixatives were decanted and the cell pellets were suspended in 500 μl of a staining solution (200 μl of RNase and 200 μl of PI), followed by incubation for 30 min at room temperature in the dark. Then, the cells were analyzed immediately by FACSCalibur flow cytometry.

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