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How to measure cell viability and cytotoxicity?

Rosaria Esposito
Tags: Cell Death, Apoptosis, Screening

A famous quote attributed to the twelfth-century philosopher Bernard De Chartres says: “We are like dwarfs on the shoulders of giants, so that we can see more than they, and things at a greater distance, not by virtue of any sharpness of sight on our part, or any physical distinction, but because we are carried high and raised up by their giant size.” In the years dedicated to scientific research, I felt the same sense of gratitude for the brilliant minds of the past. Not only for the discoveries representing the foundation of our knowledge but also for introducing techniques and methods become so essential, so common in daily laboratory practice that we easily take them for granted. One of the many examples that come to mind is represented by cell culture technology. In 1885, Wilhelm Roux managed to keep embryonic chicken cells in a saline solution for several days more than a century ago. A couple of decades later, Ross Granville Harrison could successfully grow animal tissue outside the body and introduced the main principles of tissue culture in his article describing the method for culturing nerve cells and monitoring fibers development (1). Since these pioneering works, cell culture has become more and more sophisticated. Thanks to the variety of conditions, techniques, and media currently available, we can grow and manipulate in vitro nearly every kind of cell. Cell cultures are now a fundamental instrument thanks to their multiple applications, ranging from the “classic” cell biology approaches, to genetic engineering, from producing recombinant proteins, antibodies, and vaccines, to cancer research.

The development of these techniques went, of course, in parallel with the introduction of the tools necessary for the manipulation of the model and the study of its response. In particular, in the therapeutic field, the possibility of analyzing cellular viability, toxicity, and death mechanisms is a key element in identifying potential treatments. Different parameters can be studied depending on the cell type, the preferred detection method, the specific experimental conditions, and the exact goal.


Cell viability assays

These tests can be used to assess the most suitable cell culture conditions or to analyze the effect of a given treatment/procedure on the health of the cultivated cells. They can be based on the evaluation of different factors reflecting the number of viable cells. Since the readout is the number of living cells, some assays are used for cellular proliferation analyses. The following paragraphs will briefly describe the most typical landmarks used for cell viability studies.

Metabolic activity. Probably the most intuitive parameter one can think of to understand if the cells are alive and in good conditions. It can be approached from different angles.

  • Mitochondria are the core of eukaryotic cells metabolism. They are the headquarters of cellular respiration, a complex series of enzymatic reactions eventually leading to ATP production, accompanied by the reduction of O2 molecules to H2O (see the TechNote on the role of mitochondria in cellular metabolism for further details). Mitochondrial activity is strictly related to cellular viability, and oxygen consumption rate (OCR) is, therefore, a good indicator of an active metabolism: unhealthy cells tend to have dysfunctional mitochondria and, consequently, show a lower oxygen consumption rate. One of the most accessible strategies for OCR evaluation is based on the use of phosphorescent water-soluble oxygen probes that respond to changes in the concentration of dissolved oxygen by changing its emission intensity(2). More specifically, some fluorophores exist whose fluorescence is quenched by molecular oxygen: the lower the O2 concentration, the higher the detected signal. In these assays, a drop of mineral oil is typically used to seal each sample to avoid back diffusion from atmospheric oxygen(2,3). This system has the advantage of giving direct information on cellular metabolic rate and mitochondrial activity and is directly applicable to high throughput screening analyses. However, it is extremely sensitive to relatively small variations in culture conditions, such as temperature and cell density, that must be strictly controlled for reproducible results.
  • Another way to estimate cell viability through mitochondrial activity is the quantification of ATP in the culture, as ATP levels correlate with the number of living cells. This is usually done by measuring the bioluminescence released by the reaction of ATP with luciferin catalyzed by the Luciferase enzyme (Figure 1).




Figure 1. Luciferase uses ATP to oxidize Luciferin in Oxyluciferin with the resulting production of light.



Enzo’s ApoSENSOR™ Cell viability assay kit exploits this principle in a simple and sensitive assay (detects 10-100 mammalian cells/well), compatible with automated high throughput analyses. Depending on the experimental design, the kit can be used for cell proliferation and cytotoxicity studies.

A classic group of cell viability assays, once again associated with enzymes working only in living cells, is based on reducing tetrazolium salts (e.g., MTT, XTT, WST-1, WST-8) by cellular dehydrogenases. The reaction will lead to the formation of a colored formazan product, quantified spectrophotometrically. The intensity of the colored product is directly proportional to the number of viable cells present in the culture.
MTT tetrazolium reduction assay was the first homogeneous cell viability assay developed for a 96-well format(4) and is still widely used. However, the protocol requires an additional step for formazan solubilization prior to absorbance reading, and the MTT is itself quite toxic for the cells. These drawbacks are overcome with WST-8 tetrazolium salt(5), a key component of Enzo’s Cell Counting Kit-8 (CCK-8). WST-8 reduction produces a water-soluble formazan product, eliminating the need for the solubilization step. In addition, it is more sensitive than the other tetrazolium salts (Figure 2). It is way less toxic. Once the assay is completed, the same cells can be used for other cell proliferation assays.



Figure 2. Cell proliferation assay (adherent and suspension models) using WST-8 (CCK-8) and other tetrazolium salts.



Also, in this case, the kit is often used not only to assess cell viability, but also for cell proliferation studies. For example, in a couple of recent research articles, the kit has been used to measure the proliferation of cells seeded on new biomaterials, with potential applications in regenerative medicine(6,7).

Membrane permeability: dye inclusion/ exclusion. Cell membranes (e.g., plasma membrane, nuclear envelope, etc.) have a fundamental role in the biology of the cell. Their integrity is indispensable for the cell’s correct functioning and is a good marker for monitoring cell viability.

  • Inclusion dyes can passively diffuse inside the cells where they get “stuck” once cleaved by certain families of cellular enzymes. A typical example is the Calcein acetomethoxy (AM): once inside the cell, esterases cleave the AM groups, and the calcein, now more hydrophilic, is unable to pass the plasm membrane, being trapped inside the cell. The loss of the AM group also enables calcein to bind intracellular calcium, becoming fluorescent readily. As dead cells lack cytoplasmic esterases, only viable cells will be fluorescent. Our Counting Kit-F works precisely on this principle, offering a viability/proliferation assay typically more sensitive than colorimetric assays, with a detection range from ~50 to at least 25,000 cells.
  • Exclusion dyes are fluorescent molecules that can enter and accumulate into the cell only if the plasma membrane is not intact or, in other words, when the cell is dead. Typical examples of this family of molecules are two DNA intercalating dyes: Propidium iodide (PI) and 7-amino actinomycin D (7-AAD). Exclusion dyes can be combined with other fluorescent probes to get more detailed information concerning the cells’ status or for a more subtle analysis of the death mechanism.

  • For example, each of our NUCLEAR-ID® cell viability reagents (Blue/Green, Blue/Red, Red/Green) contain a mixture of a cell-permeable nucleic acid dye (suited to stain the cells regardless of their viability status) and a cell-impermeable nucleic acid dye (suited for staining dead nuclei), with distinct fluorescent emission spectra. The staining pattern arising from the simultaneous combination of these two dyes permits the determination of live and dead cell populations in the tested sample. It is worth mentioning here that, even though these kits are typically validated in 2D cell monolayers, we do have examples of their compatibility with 3D cell models. For instance, in the two following application notes, the NUCLEAR-ID® blue/red cell viability reagent (GFP-CERTIFIED®) has been used for toxicity studies respectively on a 3D hepatocytes model human iPSC-derived and a neurosphere model:
    Finally, cells in the early apoptosis phase maintain their membrane integrity and will not be stained by exclusion dyes. For this reason, these dyes can be combined with apoptosis-specific markers (i.e., Annexin V conjugated to a fluorophore) to discriminate among the living (unlabeled), early apoptotic, late apoptotic, and necrotic cells. Check out the TechNote “How to Detect Key Features of Apoptosis?” for further details on this kind of test.



Cytotoxicity assays

Cellular toxicity assays represent the other side of the coin as they focus on detecting and quantifying the cell damage in a given condition. Their working principle is often somehow related to an impairment of cellular membranes.

Mitochondrial activity: As previously described, mitochondria produce ATP using the energy created by the membrane potential gradient. The loss of the mitochondrial membrane potential (MMP) is associated with the opening of the mitochondrial permeability transition pores, leading to the release of cytochrome C into the cytosol, which triggers other downstream events, eventually leading to apoptosis. MMP collapse detection can be the key to detecting the early phases of a cytotoxic event. Several fluorescent lipophilic cationic dyes, such as Rhod123, DiOC6, and JC-1, can be used in this context. As positively charged molecules, these dyes will accumulate within mitochondria according to membrane potential: healthy hyperpolarized mitochondria (where the matrix is more negatively charged) will accumulate more cationic dye and depolarized mitochondria (the matrix is less harmful) accumulate less dye(8). Therefore, the cytotoxic effect of a given treatment can be measured as a decrease of the fluorescent intensity associated with the cationic dye. This is the working principle of Enzo’s MITO-ID® Membrane potential cytotoxicity kit, which contains a dual-emission dye. The MITO-ID® probe accumulates as orange fluorescent aggregates in functional mitochondria and a green fluorescent monomer in the cytosol. A drop of orange fluorescence is observed as the mitochondrial function becomes increasingly compromised. This kit allows for a real-time MMP assay with a superior sensitivity compared to similar dyes (e.g., 10-fold greater than JC-1, figure 3).



Figure 3. MMP was evaluated in HeLa cells treated with CCCP using MITO-ID® dye (red) or JC-1 (blue). MMP decreases with increasing CCCP concentration, as indicated by a decrease in orange fluorescence. Improved aqueous solubility of the dye and no-wash protocol minimizes variability, leading to a higher Z-factor (> 0.9) than that obtained with JC-1.



This assay can be used as a tool to study the role of mitochondrial in drug-induced toxicity and the cellular process leading to cell death. For example, to better characterize the mechanisms underlying programmed cell death (besides apoptosis) in cancer cells, Thi Le and co-workers successfully used our kit to elucidate the role of p53 in the starvation-induced necroptosis of HCT116 colon cancer cells(9).

Enzyme leakage detection: Loss of integrity of plasma membrane due to cellular death, irremediably causes the leakage into the extracellular space of the enzymes that are usually confined inside the cell. The detection of the activity of these “escaped” enzymes is, therefore, a clear sign of cytotoxicity. In this context, the assay for lactate dehydrogenase activity (LDH) is one of the most popular solutions. LDH is a stable cytoplasmic enzyme, expressed in all cells involved in the glycolysis process. It is released into the cell culture medium due to the loss of membrane integrity following apoptosis, necrosis, or other forms of cellular damage(10).

In the typical assay procedure, LDH catalyzes the conversion of lactate to pyruvate, reducing NAD+ to NADH. The latter, in turn, reduces a tetrazolium salt into a water-soluble formazan dye, whose amount is proportional to the total LDH activity and thus to the number of damaged cells(10). Similar to the CCK-8 kit, Enzo’s LDH Cytotoxicity WST assay uses a water-soluble tetrazolium salt (WST) as a substrate (Figure 4).



Figure 4. Principle of LDH-related cytotoxicity measurement.



The two kits could be seen as two complementary assays, whose combination can provide a complete evaluation of cell health in a given experimental condition (Figure 5).



Figure 5. Complementary information was given by Cell Counting Kit-8 and the LDH Cytotoxicity WST Assay).



When thinking about cytotoxicity assays, drug-induced toxicity screenings are the most straightforward example we can think of, even though this is only one of the potential applications. For instance, a few months ago, a Korean research group used the LDH Cytotoxicity WST to measure cytotoxicity in THP-1 cells following Helicobacter pylori, in a study focusing on the protective effect of chalcone derivatives against NLRP3 inflammasome activation(11).

This general overview is, of course, far to be an exhaustive list of all the existing cell viability/cytotoxicity assays, but do not hesitate to reach out to us if you are looking for the best solution for your assays! Our Technical Support Team. We will be happy to assist!

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References:

  1. Harrison R. Observations on the living developing nerve fiber. Anat. Rec. (1907).
  2. Hynes J et al. Fluorescence-based cell viability screening assays using water-soluble oxygen probes. J. Biomol. Screen. (2003).
  3. Hynes J et al. The use of a fluorescence-based oxygen uptake assay in the analysis of cytotoxicity. Toxicol. In Vitro (2006).
  4. Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Meth. (1983).
  5. Tominaga H et al. A water-soluble tetrazolium salt useful for colorimetric cell viability assay. Analyst. Comm. (1999).
  6. Liu H et al. A simultaneous grafting/vinyl polymerization process generates a polycationic surface for enhanced antibacterial activity of bacterial cellulose. Int. J. Biol. Macromol. (2020).
  7. Choi JB et al. Fabrication and Characterization of Biodegradable Gelatin Methacrylate/Biphasic Calcium Phosphate Composite Hydrogel for Bone Tissue Engineering. Nanomaterials (2021).
  8. Perry SW et al. Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. Biotechniques (2011).
  9. Thi Le DD et al. Inhibitory role of AMP‑activated protein kinase in necroptosis of HCT116 colon cancer cells with p53 null mutation under nutrient starvation. Int. J. Oncol. (2019).
  10. Kamiloglu S et al. Guidelines for cell viability assays. Food Frontiers (2020).
  11. Choi HR et al. Interruption of Helicobacter pylori-induced NLRP3 Inflammasome Activation by Chalcone. Biomol. Ther. (2021)

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