Guava Cell Cycle Reagent for Flow Cytometry

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Cells were stained with Guava Cell-Cycle Reagent that contains the dye PI	- Cells were analysed for cell cycle after every 24, 48 and 72 h treatment with reagent

Protocol tips
-Cells were stained with Guava Cell-Cycle Reagent that contains the dye PI
Downstream tips
- Cells were analysed for cell cycle after every 24, 48 and 72 h treatment with reagent

Publication protocol

The percentage of cells in the different stages of cell-cycle was assessed by FCM and analyzed, after 24, 48 and 72 h treatment, with Guava Cell-Cycle software. Cells were fixed and permeabilized with ice-cold 70% ethanol and washed with PBS. Then, cultures were resuspended in Guava Cell-Cycle Reagent that contains the dye PI. The PI is so able to penetrate the membrane of cells, bind covalently to DNA and emit red fluorescence. In particular, cells which initially are in G0/G1 phase begins to synthesize DNA in S phase, until the complete duplication in G2/M. Therefore, the cells in G2/M phase have a double fluorescence compared to those in G0/G1 phase, while the cells in S phase have an intermediate fluorescence.

The obtained results were expressed as the percentage of cells in the different stages of the cell-cycle in treated cultures compared to that present in the control cultures.

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