Why is Viability Staining so important for flow cytometry analysis?

No matter what type of flow cytometry protocol you are using to process and analyze samples, it is very likely that you will end up with some dead cells within the population. Certain cells are just more robust than others, whereas more fragile cells may have inherent sensitivity to certain processes or reagents. Common assessed, highly labile cells include granulocytes and monocytes in whole blood, along with bone marrow resident plasma cells. In these instances, it is important to understand the optimal viability dye for any given application and panel design (Johnson, et. al. 2013).

Viability dyes enable us to selectively differentiate live and dead cells and effectively remove the dead cell population from downstream analysis. This is a critical step in any flow cytometry method since dead cells can bind conjugates non-discriminately, may display a much higher degree of autofluorescence than healthy cells, and become sticky resulting in more doublets or even clumps within the sample. All of these features can really impact the ‘cleanness’ and sensitivity of the flow cytometry assay, as well as influence how the data is interpreted. Therefore, a viability stain should be included in every flow cytometry panel and should be safe, reliable, and both time and cost-effective, and importantly, should not interfere with any test compound within the experimental system.

How do Viability Dyes Work?

Most commonly used live/dead dyes work by binding to targets present in or on the surface of dead cells, that are not normally accessible on healthy cells. There are several different classes of viability dyes, so it is important to understand their limitations and select a viability dye according to your specific sample, panel, and instrument configurations.

Figure 1. Schematic showing the principle behind the use of many viability dyes is the staining of dead cells by leveraging the increased membrane permeability and decreased integrity. Here we see how the amine-reactive dyes to bind to some of the cell surface proteins on the healthy cells, however, the binding of the dye to the intracellular proteins is several fold-higher and can be readily distinguished using flow cytometry.

DNA-Binding Viability Dyes

DNA binding dyes such as 7-AAD and propidium iodide (PI) are the most cost-effective reagents for live/dead staining. These dyes are not able to cross the cell membrane, however in the case of dead or dying cells, on which the plasma membrane is compromised, and these dyes can readily permeate and through intercalation bind to the cell DNA and generate a specific fluorescent signal. The most commonly used are summarized below. However, there are many others now commercially available – Thermo Fischer Scientific offers Sytox™, and the monomeric cyanine nucleic acid stains T-PRO™, TOTO™, and YO-PRO™, Biolegend’s product is the Hexlis-NP, and RedDot™ is offered by Biotium. These provide a range of options for excitation and emission profiles to fit almost any panel.

Table 1. Summary of the key features of the most commonly used DNA-binding viability dyes.

These dyes are simple to use and inexpensive, however, they can’t be used for fixed samples. For this type of application, almost all flow cytometrists turn to amine-reactive dyes.

Amine-Reactive Viability Dyes

Unlike the DNA binding dyes outlined previously, Amine Reactive Dyes react with all cellular proteins and since they are impermeant to intact cell membranes, they can only bind to surface proteins of live cells, whereas they can bind to the abundant intracellular proteins of dead cells. Amine Reactive dyes, therefore, stain dead cells with a much higher fluorescence. Since these molecules can withstand the harsh cross-linking reactions associated with fixation, they can be used within fixation and permeabilization protocols (Perfetto S.P, et al. 2006). They come in a range of colors allowing for compatibility with almost any staining panel, and conveniently there are now amine-reactive beads available for compensation controls that are easy to use and provide consistent results. There are some nuances to their use in that they require protein-free sample buffer and are not as easy as other viability dyes so add time to processing. Examples of commercially available amine-reactive dyes include: Biolegend’s line of Zombie™ dyes, Thermo Fischer offers eFluor™ and LIVE/DEAD™, Proteintech’s line of Phantom Dyes, BD Biosciences offers a range under the Horizon™ brand name, and Ghost Dye™ is a trademark of Tonbo Biosciences.

Figure 2. Example of LIVE/DEAD viability staining of lysed whole blood. In the sample shown, LIVE/DEAD staining is used to discriminate the live cells (96.7% of the lymphocyte population).

Enzyme-Activated Viability Dyes

An alternative to fixable dyes, Enzyme Activated Dyes that are able to stain living cells without killing them. They are used both in flow cytometry and cell imaging such as fluorescence microscopy. Typically, they are weakly or non-fluorescent until activated by some kind of cell-based enzymatic or metabolic activity. Examples include CellTrace Calcein AM that comes in a variety of colors and is membrane permeable so it can pass into cells where it is cleaved by active esterases to produce calcein, a strongly fluorescent, hydrophobic product that is well retained in the cell cytoplasm to provide staining of live cells for a few hours. This is often coupled with a DNA-binding viability dye to provide inclusion staining signals for both live and dead populations.

5-CFDA-AM is a useful dye for viability staining. Similar to Calcein, it is a target of intracellular non-specific esterase enzymes, that covert the molecule to carboxyfluorescein, a polar and non-membrane permeable fluorescent compound that accumulated in live but not dead cells.

Metabolic activity viability dyes include cell-permeable resazurin-based reagents, that permeate cells and become reduced to a red, highly fluorescent resorufin compound that is retained in the cell.

Since flow cytometry can couple viability determination with monitoring changes in cellular morphology, it is uniquely suited to track the progression of apoptosis. There are several different approaches to this, including membrane asymmetry staining, membrane permeability, and mitochondrial function assays.

Tracking Cell Viability Using Membrane Asymmetry Assays

Even though in the early stages of cell death and apoptosis cells are able to maintain plasma membrane permeability and integrity, the cell membrane changes, with the rearrangement of phospholipids and the flipping of phosphatidylserine from the intracellular surface to the extracellular surface of the plasma membrane. This phosphatidylserine can be detected using fluorescent-labelled Annexin V, a Ca2+ dependent phospholipid-binding protein (Van Engeland et. al. 1996). Annexin V staining is compatible with alcohol-free, aldehyde-based fixation, however, it can be sensitive to Ca2+ chelators such as EDTA. A more elegant approach to monitoring changes in the cell membrane involves a probe known as F2N12S (4′-N, N-diethylamino-6-(N-dodecyl-N-methyl-N-(3-sulfopropyl))ammoniomethyl-3-hydroxy flavone) that embeds into the phospholipids of the plasma membrane. This molecule has a dual-color fluorescence emission that shifts from a green emission (515-545nm) to an orange emission (564-606nm) as the apoptotic cell membrane charges are modified. The ratio of green to orange emission is used to calculate the viability percentage of the cells in the sample. Although not sensitive to calcium or proteases, F2N12S staining does require protein-free buffers.

Another way to detect apoptosis using flow cytometry is the so-called TUNEL method. One of the characteristics of apoptosis is the degradation of the chromosomal DNA and the generation of DNA fragments ranging from 180-200 base pairs in length. The TUNEL methods utilize the enzyme Terminal Deoxynucleotidyl Transferase (TdT) incorporate fluorescent-labelled dUTP onto the 3-OH end of these fragments, therefore thereby labelling the apoptotic cells for flow cytometry detection.

Mitochondrial Targeting Dyes:

These fall into two categories, Structural Dyes, and Functional Dyes.

Structural Dyes such as MitoD and Nonyl Acridine Orange (NAO) stain mitochondria through binding to the cardiolipin on the inner mitochondrial membrane, whereas MitoTracker Green FM labels the mitochondrial through binding to the abundant free thiol groups of cysteine of the mitochondrial proteins. All three of these dyes are typically used to look at the abundance of mitochondria in a cell- or the mitochondrial mass, or more specifically the inner mitochondrial quantity. However, it is important to note that this varies considerably between different cell types within the same species, as well as between the same cell types that are in different metabolic states.

Functional Mitochondrial dyes are useful for the assessment of early apoptosis, during which the disruption of the mitochondrial membranes disrupt the membrane potential that is central to mitochondrial health and function. Generally, these are highly membrane-permeable, cationic fluorescent dyes that are attracted to the highly negative electrical potential across the inner mitochondrial membrane. They, therefore, accumulate in the mitochondrial matric of living cells. There are three common families of these functional dyes:

Rhodamine dyes: rhodamine 123 (Rh123), tetramethylrhodamine ethyl ester (TMRE) and tetramethylrhodamine methyl ester (TMRM),

Carbocyanine dyes: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine (JC-1), 3,3′-dimethyl-α-naphthoxacarbocyanine iodide (JC-9) and 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)),

Rosamine dyes: orange, red, and infra-red-fluorescent MitoTrackers

Each of these classes come in different options that cover a range of emission spectra to fit into even complex panel. It is important to use the lowest amounts of these dyes since staining with mitochondrial-specific probes induces inhibition of respiration (Cottet-Rousselle et. al. 2011). Below is an example of apoptosis tracking in a population of ThP-1 cells using MitoTracker Red.

Figure 3. THP-1 cells untreated (left) and 5 h after 20 μM etoposide treatment (right) stained with MitoTracker Red (50 nM; mitochondria) and Hoechst 33258 (5 μg/ml−1; plasma membrane integrity). Viable cells with intact mitochondrial membrane potential are located in the lower-right quadrant. Early apoptotic cells exhibiting ΔΨm dissipation and intact plasma membrane are located in the lower-left quadrant. Late apoptotic (or necrotic) cells with depolarized mitochondria and permeabilized plasma membrane are located in the upper-left quadrant.

So before selecting a viability dye for your panel, here are some key considerations:

Flow Cytometer as a method for which dead cells can really impact the dynamic range of detection and make it difficult to distinguish truly positive signals. It is therefore highly recommended that some form of viability stain be incorporated into every one of your panels. Whenever selecting a viability dye there are some key considerations:

• Is your sample fixed or unfixed?
• Consider all of the sample processing steps- where should a viability stain be applied? and how stable that staining is overtime? For fixed samples, it is important to establish if viability staining is introduced before or after antibody staining.
• Identify a viability dye that is compatible with the fluorophores in the panel- if you need some help with this, Fluorofinder.com is a great resource.
• Ensure that your instrument configuration (laser and bandpass filters) is compatible with the use of that viability dye in that channel.
• Consider the type and origin of the cells that are being analyzed, since not all cells stain the same way.
• When analyzing samples with a high % of dead cells, it might be worth using two viability dyes, that together provide inclusive staining of both live and dead populations.
• For flow cytometry panels that require validation, make sure the viability staining for your target cell populations is optimized and establish the appropriate positive and negative controls.

With so many options for viability dyes in all different excitation and emission properties, researchers now have great flexibility in incorporating viability dyes into the most complex, multi-parameter flow cytometry panels. Careful consideration of the viability dye selection and its’ inclusion into the sample processing will ultimately help generate cleaner and more robust flow cytometry data sets.

References

• Johnson, S., Nguyen, V., & Coder, D. (2013). Assessment of cell viability. Current Protocols in Cytometry, 64(1), 9.2.1– 9.2.26
• Perfetto S.P, et al. (2006) Amine reactive dyes: An effective tool to discriminate live and dead cells in polychromatic flow cytometry. Journal of Immunological Methods, 313, Issue 1-2, 30, pp 199-208. https://doi.org/10.1016/j.jim.2006.04.007
• Van Engeland, M., Ramaekers, F. C., Schutte, B., & Reutelingsperger, C. P. (1996). A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture. Cytometry: The Journal of the International Society for Analytical Cytology, 24(2), 131– 139.
• Cottet-Rousselle et. al. (2011) Cytometric assessment of mitochondria using fluorescent probes. Cytometry Part A Vol. 79A Issue 6, pp 405-425.