Flow cytometry analysis only provides usable data if you analyze living cells. In both preclinical and clinical studies, including a live/dead stain in flow cytometry panels is essential for discriminating live from dead cells in order to analyze only live cells. Live/dead stains are also called viability dyes, and different types of dyes use different chemistry to stain live or dead cells. Consider these factors when including a live/dead stain in flow cytometry panels.
- What kind of flow cytometer are you using? The flow cytometer dictates which live/dead stains can be detected based on the excitation wavelengths emitted by the lasers and emission wavelengths that can be detected. Be sure to use a live/dead dye that can be detected on the flow cytometer you plan to use.
- How does a live/dead dye fit with my staining panel? The number and types of stains you can include in your staining panel depends on your flow cytometer. Also, inclusion of a live/dead dye may mean that an existing fluorescently labeled antibody may need to be replaced if it has an overlapping emission wavelength.
- What is the chemistry behind the dye? The original class of live/dead dyes, like propidium iodide, bind to double-stranded DNA, which is only accessible in dead cells with damaged cell membranes. Alternatively, protein binding dyes can bind primary amines on the surface of live and dead cells but can also gain access to intracellular amines in dead cells with damaged membranes, which results in much brighter staining. Consider which live/dead staining method works best with your protocol and carry out pilot studies to evaluate different dyes.
Live/dead stains in flow cytometry panels are essential to getting reliable data, so consider working with a professional in flow cytometry services to have confidence in your staining panel and the overall quality of your results.
