Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
A basic but crucial task facing researchers who analyze live-cell populations is to assess their viability. In its simplest form, this involves counting live and dead cells. But researchers need tools to distinguish between the two, as well as reliable means of counting them swiftly. Enter flow cytometry.
Excluding dead cells
Many methods exist for making dead or dying cells detectable by flow cytometry. Although all fall under the heading of “cell-viability tools,” they don’t all work the same way.
“‘Viability’ is a very loosely defined term that could potentially be taken to mean any of [several] functions in the context of analysis by flow cytometry,” says Caleb Hopkins, product manager in protein assays at Sigma-Aldrich.
Probably the most common and simplest approach to viability tests cell-membrane integrity via dye exclusion, Hopkins says. In this method, researchers add to the extracellular milieu a dye that stains cells with compromised cell membranes but is excluded from cells with healthy, intact cell membranes. The dyes, which stain nuclei by binding to double-stranded nucleic acids, include propidium iodide (PI), 7-amino-actinomycin D (7-AAD), ethidium homodimer, TO-PRO®-3 (a carbocyanine-based dye from Thermo Fisher Scientific) and 4',6-diamidino-2-phenylindole (DAPI).
BD Biosciences’ BD Via-Probe™ Cell Viability Solution contains 7-AAD; the BD™ Cell Viability Kit uses thiazole orange to stain all cells and PI to stain dead cells exclusively. Other options include Thermo Fisher Scientific's SYTOX® Dead Cell Stains and Bio-Rad Laboratories’ ReadiDrop™ 7-AAD Cell Viability Dye and ReadiDrop Propidium Iodide Cell Viability Assay.
Enzo Life Sciences offers two cell-viability kits, the Nuclear-ID® Blue/Green and the Nuclear-ID Blue/Red Cell Viability Reagents. Using two nuclear stains to distinguish between live and dead cells, each incorporates a proprietary asymmetric cyanine dye that is excluded from healthy cells but stains the DNA of dead cells (green or red, depending on the reagent). This is paired with a cell-permeable, blue-fluorescent dye that binds nucleic acids, staining the nuclei of viable cells. The Nuclear-ID® Blue/Red version is suitable for use with cells expressing GFP. Another tool from Enzo Life Sciences, the Cyto-ID® Autophagy Detection Kit, detects cells undergoing autophagic death, says Courtney Noah, senior marketing manager at Enzo. “The Cyto-ID® Autophagy Detection reagent is a proprietary green dye that stains autophagic vacuoles, thus enabling monitoring of autophagic flux in live cells.”
Another class of reagents specifically stains apoptotic cells. “In the early stage of apoptosis, cells lose their phospholipids’ membrane asymmetry and expose phosphatidylserine on the cell surface,” says Kazuyuki Atarashi, global program manager for research products at MBL International. This can be detected using fluorescently tagged annexin V, which binds to phosphatidylserine.
Atarashi says MBL’s MEBCYTO® Apoptosis Kit uses annexin V labeled with fluorescein isothiocyanate (FITC) to detect early apoptosis and PI to detect later stages of apoptosis, when the leaky membrane allows PI to reach the nucleus and stain DNA itself. Related tools include Enzo Life Sciences’ annexin-based GFP-Certified® Apoptosis/Necrosis detection kit, BD Biosciences’ Annexin V-FITC Apoptosis Detection Kit II, eBioscience/Affymetrix’s Annexin V FITC Apoptosis Detection Kit and R&D Systems’ TACS Annexin V-FITC Apoptosis Detection Kit.
Irreversible viability dyes are yet another type of reagent that may prove useful for researchers who plan to tag multiple targets with different fluorophores. Sometimes called ‘fixation dyes,’ because they are suitable for fixation conditions, these dyes can withstand the many washes and incubations necessary for subsequent tagging with antibodies and fluorophores. This is useful when staining dead cells before staining for other targets for flow cytometry. Fixable dyes are available from several companies and include Molecular Probes® LIVE/DEAD® Fixable Dead Cell Stain Kits from Thermo Fisher Scientific (Life Technologies), eFluor® Fixable Viability Dyes from eBioscience, BD Horizon™ Fixable Viability Stains from BD Biosciences and VivaFix™ cell-viability assays from Bio-Rad.
Signs of life
Many vendors offer cell-viability kits that include a pair of dyes—one for dead cells (usually for dye exclusion, as described above) and one for live, viable cells. Several methods exist for staining live cells; most are based on a particular cell function, such as intracellular esterase activity, asymmetry of membrane lipids or proliferative capacity. The significance of these functions is they represent observable processes that researchers can assay as indicators of cell health.
Some such assays make use of calcein-acetoxymethyl compounds (calcein AM, available in several different colors) that can permeate healthy cell membranes. Within the cytoplasm, cellular esterases cleave the AM ester group, rendering the calcein dye both membrane-impermeable and fluorescent. Examples include Thermo Fisher Scientific's LIVE/DEAD® Viability/Cytotoxicity Kit, Sigma-Aldrich’s Live/Dead Cell Double Staining Kit and R&D Systems’ Calcein AM Cell Viability Assay.
Another type of functional test for live cells uses mitochondrial dyes. These will enter healthy mitochondria that have a mitochondrial membrane potential and remain trapped within, where they fluoresce. Dead cells, or unhealthy cells with compromised cell membranes, lack a normal mitochondrial membrane potential, and so the dye is excluded. R&D Systems’ DePsipher Mitochondrial Potential Kit works this way.
Viable cells may be alive, but that doesn’t necessarily mean they are healthy enough to divide and proliferate. Cell-tracking dyes can be used to label cells and are then washed away. Then, by measuring the rate of “dye dilution” that occurs as cell division proceeds, researchers can estimate cell proliferation. Enzo Life Sciences’ Cyto-ID® Green and Cyto-ID® Red long-term cell-tracer kits operate by this principle. According to Noah, “Dye-labeled and unlabeled cell populations can be analyzed by flow cytometry, since no transfer of fluorescence to adjacent cells [is] observed after a prolonged 96-hour incubation period and is passed to daughter cells upon mitosis.” Similar tools include Bio-Rad’s CytoTrack™ Cell Proliferation Assays and Sigma-Aldrich’s lipophilic cell-tracking dyes PKH26, PKH67 and CellVue® Claret.
Choosing colors
The plethora of fluorescent tools available for assessing cell viability is both a blessing and a curse. On one hand, it enables researchers to assess viability while detecting fluorescence from several other target proteins simultaneously. The number of colors that can be read at once is limited only by the number and type of laser lines in your flow cytometer. But on the other hand, choosing which colors to use requires careful consideration to avoid spectral overlap with the dyes used to assess viability. “When you’re looking at flow cytometry, it’s kind of a balance point of what colors you need to use on specific antibodies for the detection of receptors or protein epitopes, but in combination with the appropriate dyes that don’t cause problems with detection,” says Bob Balderas, vice president of research and development at BD Biosciences.
Balderas advises researchers to consider several important questions when choosing colors for dyes and fluorescently labeled antibodies in flow cytometry. Your flow cytometer’s laser configuration is one—obviously, the more color channels you have, the more flexibility you have in experimental design. Another is the range of available colors.
Balderas says matching a fluorophore to an antibody “has a lot to do with the density of the antigen receptor or the co-expression of the receptor that you’re checking on the surface of the cell.” However, receptors are expressed at various amounts, and your choices should optimize all signals simultaneously.
Balderas says a “systematic approach of developing a multicolored cocktail” can help researchers choose viability dyes that can be used successfully with different fluorophores and lasers. But don’t neglect sample quality. “If you put a cell that’s compromised into an experiment, then there’s always an opportunity for compromised data,” he says. “At the end of the day, the true value of great data is the quality of the sample.”