Drug treatments, genetic manipulations and simply handling cells can all have an effect on the health of your samples. It is important to take these effects into consideration when analyzing your flow cytometry experiment. Dead cells can skew your results by nonspecifically taking up monoclonal antibody (mAb) conjugates, contributing to cellular autofluorescence (background), and causing cell aggregation. This is especially problematic when you are measuring low-expression antigens or counting rare cell populations. Viability dyes enable you to distinguish these two cell populations (live and dead) in your analysis. They generally fall into a few different categories and you can choose from a range of color options. (see Table 1).
Nonviable cells
The most common types of viability dyes are the classic dyes and the protein binding dyes, which predominantly stain dead cells. Both are dependent on cell membrane integrity and require compromised cell membranes to enter the cell. The classic dyes bind to nucleic acids (DNA and in some cases RNA), while the protein binding dyes react with amines in the cytoplasm and on the cell surface. Protein binding dyes are most appropriate for fixed cells. Derek Davies, head of the Flow Cytometry Laboratory at the Francis Crick Institute, United Kingdom says “The range of dyes and the number of manufacturers producing them has increased specifically in the amine-reactive dyes – most of the major companies in the flow field will now produce a range of dyes excited with different wavelengths and emitting in different ranges. This increases our choice further.” He also warns “If sorting and using an aminereactive viability dye, be aware that live cells are also stained, just at a lower level than dead ones, and the dye will be on your sorted cell.”
Viable cells
Alternatively, you may prefer to stain viable cells in your samples using dyes such as Calcein AM (multiple colors available) or esterase substrate carboxyfluorescein diacetate (cFDA). These dyes are indicators of esterase activity and cell membrane permeability. With Calcein AM, intracellular esterases cleave the acetoxymethyl ester (AM), activating the dye. The fluorescent Calcein is retained within cells possessing intact cell membranes. Other choices include resazurin and its lipophilic analog, dodecylresazurin (C12-resazurin), both of which are used for staining metabolically active cells. Unlike nonviable cells that lose their metabolic activity, viable cells are able to reduce nonfluorescent resazurin to red-fluorescent resorufin. Pairing viability dyes together can improve separation and allow you to better segment your cell populations. You can either combine them yourself or purchase one of the commercially available kits. Derek advises “As with all probes we should check that they have no effect on viability and titrate them as we would other dyes – if we are simply excluding dead cells, then try to use at the lowest concentration that still allows easy identification of the cells of interest.” One of the biggest challenges used to be determining what dyes would work with your specific lasers and optical filters on your cytometer. Since FluoroFinder incorporates cytometer configurations into the panel design software, you can automatically view all the available and optimal viability dyes for your cytometer. Furthermore, you can view each dye’s excitation and emission profile and determine how well it will fit with the rest of your panel. Adding viability dyes can reduce uncertainty in your results. The wide range of available viability dyes spanning the color spectrum and the ease with which you can accurately add them to your panel make it fast and simple to add these critical dyes to your experiments.
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