Cell viability assessment is a crucial application in flow cytometry, commonly used to evaluate the health and functionality of cells. This assessment typically involves two primary categories of dyes, each with distinct mechanisms of action.
1. Membrane-Permeable Dyes
The first category includes dyes that enter cells through compromised membranes, allowing them to bind to intracellular components. A well-known example is propidium iodide (PI), which is excluded from viable cells due to intact membranes but can penetrate and stain cells with damaged membranes. Once inside, PI binds to nucleic acids, fluorescing upon excitation. This characteristic makes it a valuable tool for identifying dead or dying cells, as only those with compromised membranes will fluoresce.
2. Enzyme-Activated Dyes
The second category consists of vital dyes that can traverse the membranes of healthy cells. These dyes undergo enzymatic conversions within the cell, transforming into fluorescent molecules. For instance, calcein AM is a common enzyme-activated dye that is non-fluorescent in its initial form but becomes fluorescent upon hydrolysis by intracellular esterases. This property enables the detection of live cells, as the dye remains within viable cells and emits fluorescence.
If the cell membrane is damaged, the enzyme-activated dyes can leak out, resulting in the loss of fluorescence. This property is especially useful for differentiating between live and dead cells based on membrane integrity.
In situations where cell fixation is required, it is essential to use fixable dyes. These dyes are designed to maintain their staining properties even after the fixation process, allowing for accurate analysis of cellular components while preserving the integrity of the dye signal. Fixable dyes are critical in applications such as immunophenotyping, where the preservation of cellular markers post-fixation is necessary for reliable results.
In summary, the assessment of cell viability using flow cytometry involves a combination of membrane-permeable and enzyme-activated dyes, each providing unique insights into cell health. Proper selection of dyes, especially in the context of fixation, is vital for the accuracy and reliability of the results obtained in cellular analysis.