PI/RNASE Solution

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cells (1×106 cells/well) were incubated with vehicle or 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate at their respectively IC50.	- After 24 h of incubation cells were treated with the protocol

Upstream tips
- Cells (1×106 cells/well) were incubated with vehicle or 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate at their respectively IC50.
Protocol tips
- After 24 h of incubation cells were treated with the protocol

Publication protocol

The compound's effect on cell cycle was investigated by PI/RNase solution kit (Immunostep, Salamanca, Spain). Cells (1×106 cells/well) were incubated with vehicle or 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate at their respectively IC50. After 24 h of incubation, cells were harvested and cell cycle analysis was assessed according to the kit protocol. Briefly, cells were harvested, washed with PBS and centrifuged for 5 min at 300 g. The supernatant was removed and cells were fixed with 70% ethanol for 30 min at 4°. Cells were washed once with PBS supplemented with 2% bovine albumin, centrifuged for 5 min at 300 g and then the supernatant was removed. To the cell pellet it was added 500 µl of PI/RNase solution followed by 15 min incubation at room temperature. Finally, the analysis was performed by flow cytometry as previously described.

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