The propidium iodide (PI) cell cycle protocol is a common flow cytometry application used to analyze the cell cycle progression of a population of cells. This protocol involves staining cells with PI, a fluorescent DNA binding dye that intercalates into double-stranded DNA, and then measuring the fluorescence intensity of the cells using flow cytometry.
Here is a general protocol for the propidium iodide cell cycle analysis:
Harvest cells
Harvest cells using standard cell culture techniques and suspend them in a single-cell suspension.
Fix cells
Fix cells with 70% ethanol or a similar fixative solution to stabilize the cell membranes and prevent DNA degradation.
Stain cells with PI
Resuspend cells in a buffer containing PI and RNase. PI binds to the DNA in cells, and RNase helps to remove RNA from the cells to prevent interference with the analysis. Incubate cells for at least 30 minutes at 37°C in the dark.
Analyze cells using flow cytometry
Analyze cells using a flow cytometer with excitation at 488 nm and emission at 585 nm. The fluorescence intensity of the stained cells is proportional to the DNA content of the cells. The flow cytometer generates a histogram of fluorescence intensity that represents the distribution of cells in different phases of the cell cycle (G0/G1, S, and G2/M).
Data analysis
Analyze the flow cytometry data using specialized software to calculate the percentage of cells in each phase of the cell cycle. The software can also generate graphs and other visual representations of the data.
Overall, the propidium iodide cell cycle protocol is a widely used technique for analyzing the cell cycle progression of a population of cells using flow cytometry.
Learn about Sapio Sciences’ flow cytometry data analysis tool here.