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.
Cell biologists have much to be grateful for when it comes to modern technology. Previous generations assessed the viability of their cell cultures by counting healthy and dead cells one by one under a microscope. Today’s fluorescent cell-viability assays spare us this tedious and painstaking work. The ability to monitor cell viability is particularly crucial in predictive toxicology studies, which use measurements of live and dead cells, sometimes compared to computerized cell models, to predict the toxicity of drug compounds. This can save considerable time and money in the drug-development process by helping to eliminate toxic compounds earlier in the drug-discovery pipeline. But cell-viability assays are also used every day in research labs around the world to assess the health of cell cultures.
Most cell-viability assays work according to the general principle of making live and dead cells easily distinguishable from each other so they can be counted. Some assays stain live cells in a manner that is impossible with dead cells. For example, live cells have intact plasma membranes and contain properly functioning enzymes such as proteases, esterases and dehydrogenases. Thus, if an assay relies on the presence of such features to generate fluorescence, you can be confident that the fluorescent signal can be correlated with live cells. Conversely, dead cells lack working enzymes and an intact plasma membrane. Some cell-viability assays use separate stains for live and dead cells (see below). This is useful, because in some situations reagents can bind to dead cells nonspecifically, which can lead to false positive signals. Most assays today are adaptable to different formats, such as fluorescence microscopy, flow cytometry and microplate readers; however, some assays are particularly suited to one format.
Assays based on protease activity
Assays that measure protease activity usually rely on a constitutive protease contained in live cells. A substrate that is lypophilic enough to cross intact cell membranes is added to the culture medium. Upon entering the cell, the substrate is cleaved by proteases, which causes it to fluoresce. The fluorescent signal generated by protease activity thus represents the number of live cells present. This type of assay is compatible with all formats and can also be multiplexed with other assays or geared up to higher throughput when performed in plates or wells.
Assays based on membrane permeability
Assays based on membrane permeability rely on healthy cell membranes of viable cells. For example, some fluorescent dyes bind to free amine groups on proteins. If the dyes are membrane-impermeant, they remain excluded from live cells but enter the intracellular remains of dead cells and bind to residual proteins. Live cells, therefore, show little fluorescent labeling, but dead cells fluoresce brightly. This type of assay can be used in any format and is also particularly useful for excluding nonviable cells when separating cells by flow cytometry, such as with fluorescence activated cell sorting (FACS).
Another important and commonly used example of dyes that rely on membrane permeability is the nuclear dye propidium iodide, which fluoresces red when it binds to DNA. Propidium iodide is a reliable marker of dead cells, because it cannot cross a cell’s plasma membrane. Thus live cells can exclude the dye, but dead cells cannot.
Assays based on redox sensitivity
Though these assays also rely on enzymatic activity and intact cell membranes of viable cells, they stand out because they use redox-sensitive compounds. Several use variations on a tetrazolium-based compound, such as XXT. When XXT is added to culture media, its solubilized tetrazolium salt enters the cell and is cleaved to become a Formazan dye that fluoresces orange—but only in live cells that still retain functioning enzymes. MTT, a similar compound of a yellow color, is reduced in live cells to become a purple Formazan dye (in fact, in an insoluble crystal form). The redox-sensitive dye Resazurin, similarly, begins as a nonfluorescent blue dye but changes to a highly-fluorescent pink color when reduced by dehydrogenase enzymes in viable cells. These compounds are all very amenable to microplate reader formats.
Two-color assays
Assays for both live and dead cells are usually based on the use of two fluorescent dyes that label the live and dead cells in different ways. Dead cells (or rather, their nuclei) are indicated by the red fluorescent dye propidium iodide, as described above. Live cells are typically labeled using the green fluorescent dye calcein. When the lipophilic (and nonfluorescent) compound calcein-AM is added to media, it crosses the plasma membranes of cells, where intracellular esterases cleave it to produce fluorescent calcein. Calcein alone cannot cross the plasma membrane, so the label is trapped within live cells. Fluorescent signals from both live and dead cells can be seen simultaneously at an excitation wavelength of 490 nm. Two-color, live-dead cell assays can be used in all assay formats, including flow cytometry, fluorescence microscopy and fluorescence microplate readers.
Today’s high content imaging and analysis systems are powerhouses of cell-viability monitoring, capable of assessing a multitude of parameters simultaneously—such as cell number, nuclear status, mitochondrial integrity, calcium homeostasis, cell viability and apoptosis. Systems like these, which can fly through measurements using 384-well plates, can generate enormous amounts of detailed information about your cells. If you need to know more than simply how many cells are alive or dead, such systems may help you to see into the mysteries of the intracellular world.
The image at the top of the page is from Life Technologies.