Olivia L. May,P.h.D.

Measurements for cell viability are used to evaluate the death or life of cancerous cells, the rejection of implanted organs, or the effectiveness of a drug candidate. Cell viability assays are also often useful to determine optimal growth conditions of cell populations maintained in culture. Cayman offers a group of related assays that can be used to assess proliferative activity, cell viability, metabolic activity, cell cycle phase, cell toxicity, or apoptosis. Together, the information derived from these assays can indicate whether a cell population that has been exposed to an experimental stimulus is healthy or dying, actively dividing or in stasis, or has committed to an apoptotic pathway. Like a detective searching for clues, in order to gather proper evidence, it’s important to ask the right questions, or in a researcher’s case, select the most appropriate assay. With the variety of assay approaches available, this task can seem daunting. Luckily, features of the very cells themselves provide signs of proliferative activity and capability. Here’s how to read them:

The most direct means of measuring cell proliferation, a determination of the number of actively dividing cells, is to count the number of cells present. Cell viability, defined as the number of healthy cells in a sample, determines the amount of cells (regardless of phase around the cell cycle) that are living or dead, based on a total cell sample. While a basic cell count is a direct measure of proliferation and viability, measurements of DNA content or metabolic activity are correlates that can offer more information about the physical condition and cell cycle stage.

Plasma Membrane Integrity

Assessing cell membrane integrity is one of the most common and straightforward ways to measure cell viability and assess cytotoxic consequences. Compounds that have cytotoxic effects often compromise cell membrane integrity and induce necrosis. Dyes, such as propidium iodide and 7-AAD, are normally excluded from the inside of healthy cells; however, if the cell membrane has been compromised, they freely cross the membrane and stain intracellular components. This method distinguishes healthy cells with uncompromised membrane integrity (unlabeled) from non-healthy ones (colored). These dyes are included in several Cayman kits, including the 7-AAD/CFSE Cell-Mediated Cytotoxicity Assay Kit, Annexin V FITC Assay Kit, and Glucose Uptake Cell-Based Assay Kit. These kits each use membrane permeability as a single measure of cellular viability within the context of a specific system, and will be described in more detail below.

With the increase in immunotherapy techniques to treat cancers, cell-mediated cytotoxicity is an important parameter to measure. Cayman’s 7-AAD/CFSE Cell-Mediated Cytotoxicity Assay Kit is a user-friendly, rapid way to assess the cytotoxicity of effector cells against the chosen target. 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) is employed to stably label target cells. Once targets and effector cells are mixed, the target cells can be identified by this CFSE label and death can be assessed by 7-AAD positivity. Alternatively, membrane integrity can be assessed by monitoring the passage of substances that are normally sequestered inside cells to the extracellular environment. One commonly measured molecule is lactate dehydrogenase (LDH), a soluble cytosolic enzyme that is released into the culture medium following the loss of membrane integrity. Cayman’s LDH Cytotoxicity Assay Kit detects this cytosolic enzyme released as a result of membrane breakdown during experimental treatment-induced cell death.

Mitochondrial Function and Apoptosis

While the most prominent role for mitochondria is the production of ATP, the major source of cellular energy, these power generators have been linked to a full gamut of cellular activities. Other important roles of mitochondria include cell signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Given these collective functions, assessing mitochondrial function gives a fair indication of cell health. Cayman has recently developed a series of Mitocheck^® ETC Activity Assay Kits to measure the activity of each individual complex of the electron transport chain. Often mitochondrial damage accompanies cytotoxic effects. Also, mitochondria can trigger apoptosis by disrupting electron transport, oxidative phosphorylation, and ATP production, by releasing proteins that activate caspase family proteases, or by altering cellular oxidation-reduction potential. JC-1 is a carbocyanine liquid crystal-forming dye used to analyze mitochondrial membrane potential. Cayman’s JC-1 Mitochondrial Membrane Potential Assay Kit reliably probes mitochondrial membrane potential changes. Cayman’s Caspase-3 Fluorescence Assay Kit detects mitochondrial-triggered apoptosis by identifying activation of the specific apoptotic marker, caspase-3.

Additional assay kits available from Cayman can be used to detect morphological changes related to distinct time points during the apoptotic process. The Annexin V FITC Assay Kit reveals changes in plasma membrane asymmetry that signify early stage apoptosis. Phospholipids are asymmetrically distributed at the plasma membrane with phosphatidylserine (PS) predominantly observed on the inner surface facing the cytosol. In the early phases of apoptosis, although the cell membrane remains intact, this asymmetry is disrupted, thereby exposing PS to the outer layer of the membrane. Annexin V (in the presence of Ca2+) preferentially binds to negatively charged phospholipids like PS. While the critical degradation process of autophagy most often acts to promote cell survival in response to stress, it can also promote cell death. Cayman’s Autophagy/Cytotoxicity Dual Staining Kit employs the autofluorescent probe monodansylcadaverine as well as propidium iodide to detect both autophagic vacuoles and dead cells.

Cellular Proliferation

Many researchers accept metabolic activity as an alternative for measuring proliferation. While changing cell number is an absolute measure of cell proliferation, metabolic activity is more a measure of cell health. Combining these assays can yield a more detailed view of cell activity. In cancer drug discovery, for example, the goal may not be to cause cell death but simply to knock down the metabolic and proliferative activity of a cell with cell stasis being the desired outcome.

A classic approach to assessing metabolic activity involves the use of tetrazolium salts that are cleaved by metabolically active cells to form colored, water-insoluble (MTT) or water-soluble (XTT, WST-1, and WST-8) formazan salts that can be measured by absorbance (Figure 1). The amount of formazan dye produced is directly proportional to the number of metabolically active cells and indicates the reducing potential of the cell. Cayman’s cell proliferation assays, MTT, XTT, WST-1, and WST-8, employ this method as a tool for studying the induction and inhibition of cell proliferation in any in vitro model. The MTT assay has a long-held reputation as the conventional cell proliferation assay and carries with it a strong body of literature support. However, MTT is cleaved to a water-insoluble formazan crystal that must be solubilized before reading on the spectrophotometer, which leads this assay to have a relatively long protocol.

Figure 1. Extracellular reduction of WST-8 to its formazan dye by intracellular NADH or NADPH is facilitated by an electron mediator, 1-Methoxy PMS.

XTT, WST-1, and WST-8 produce aqueous-soluble formazan products, eliminating the need for a solubilization step, which facilitates assay optimization. In particular, WST-8 is more stable and less cytotoxic compared to other tetrazolium salts, making it especially useful for longer incubation periods. Furthermore, the detection sensitivity at a high cell density of WST-8 is higher than that of other tetrazolium salts. Conversely, XTT is more sensitive to lower cell numbers, and thus may be more useful in less metabolically active cells, or in applications where the cell number is limiting.

In comparison to traditional, radioactive [3H]-thymidine or nonradioactive 5’-bromo-2’deoxy-uridine assays, MTT, XTT, WST-1, and WST-8 have the advantage of ease of use. However, it is important to be aware of the caveats of these assays. Since they measure the metabolic activity of cells, it will not be possible to distinguish cytotoxicity from cytostasis using solely a tetrazolium salt assay. It may be useful, then to combine these assays with others, such as the LDH Cytotoxicity Assay Kit, to gain more information about how selected compounds are affecting cells.

Healthy, intact cells can also be fixed, permeabilized, and labeled to study cell cycle progression. Propidium iodide, used in Cayman’s Cell Cycle Phase Determination Assay Kit, labels DNA in cells undergoing various phases of the cell cycle. Because the dye directly intercalates with the base pairs on a DNA strand, its fluorescence intensity is directly proportional to the DNA content of the cell. This can reliably distinguish cells in G0/G1 versus S versus G2/M. Further, the CFSE Cell Division Assay Kit employs a fluorescent dye that is capable of diffusing through the plasma membranes of healthy cells. As the cell divides, the fluorescence is retained in successive generations, though sequentially decreases by half with each division of cells. This assay kit is especially useful for immunologists studying lymphocytes, where proliferation can be a proxy for activation.

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