How can I tell the difference between alive and dead cells during flow cytometry?


You can use fluorescent labels to identify dead cells, to identify live cells, or combine them both in a two parameter assay.

The most common way to identify dead cells is using a cell-impermeant DNA binding dye, such as propidium iodide or a dye from the STYOX series. A healthy living cell has an intact cell membrane and will act as a barrier to the dye so it cannot enter the cell. A dead cell has a compromised cell membrane, and it will allow the dye into the cell where it will bind to the DNA and become fluorescent. The dead cells therefore will be positive and the live cells will be negative.

If you need to fix your cells, there is another option, using the LIVE/DEAD fixable stains, also known as amine reactive dyes. This also works using the principle of cell membrane integrity, where amines on the outside of a living cell with react with the amine-reactive dye and have a dim fluorescence, while a dead cell with a compromised membrane will allow the dye into the cell where it will react with amines throughout the cell resulting in a bright fluorescence. Unlike the DNA-binding dyes, the amine reactive dyes are fixable. You can label you cells with the LIVE/DEAD Fixable stain, and then fix the cells, and the distinction of live and dead cells will be maintained.

You may want to also look at a metabolic indicator, such as Calcein AM or C12-resazurin, so called vitality indicators. A metabolic label and a dead cell stain can be combined for a more complete look at cell health.

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