Cytotoxicity assays: investigating a ‘toxic environment’ to your cells

What is a cytotoxicity assay?

Toxicity of exogenous physical, biological, or chemical agents can be investigated on an organ- or whole-body level during animal experiments [1]. However, these experiments come with numerous ethical objections and lack the possibility to investigate human cells [1-3]. Therefore, in vitro cytotoxicity experiments are performed, replacing the animal experiments (3R: refine, reduce, replace) and enabling research on human cells. The term “cytotoxicity” refers to toxicity to cells [4], thereby investigating whether the exogenous agents do not interfere with healthy cell functionality and do not cause apoptotic or necrotic cell death [3-5]. This information is valuable for a number of research areas and experimental applications. For example, the suitability of bioactive compounds – like drugs and nanoparticles – as clinical therapies is based on the cytotoxicity of the compounds to healthy and diseased cells, where the main cytotoxic effect should be exclusively observed in the diseased cells [4]. Furthermore, the biocompatibility of medical devices in direct/indirect contact with the human body is essential for the proper functioning of these devices. One could think of biomaterials, prostheses, medical imaging systems, and systems involving radiation [6]. Besides that, in biomedical sciences, models of cells and tissues are often used, which require sufficient physiological relevance. Here, cytotoxicity screening experiments can be performed for cell culture quality control, to investigate fundamental cell and tissue processes, for disease modeling, and for infection and immunology research [1, 2]. Moreover, in toxicology research, environmental factors (e.g. pollution, micro-/nanoplastics) can be related to diseases when investigating the cytotoxicity of these factors [2, 3]. The abundant use and fundamental role of cytotoxicity assays in cell research require optimal execution of the corresponding experiments, to obtain the desired results. Therefore, this article provides a summary of the basic principles, approaches, and methods of in vitro cytotoxicity assays.


How to test cytotoxicity: methods

Exposure of cells to the exogenous agent

To be able to investigate the cytotoxicity effect of the exogenous agent on the cells’ health, the cells have to be exposed to the agent. There are three main strategies for this, where the most suitable strategy for an experiment depends on the type of sample and the research question [6].

(1) In a direct contact setup, the agent is directly added to the cell culture. This strategy provides high sensitivity since already the smallest amount of agent can influence the cells, without any preceding dilution processes. It is applied for the purest and well-defined samples: physical or biological agents as well as chemical agents with a known molecular composition (e.g. drugs) [6].

(2) In an indirect contact setup, the investigated sample is placed on top of a disc through which toxins leach onto the cells. Since the disc often consists of agar, this is also named the “agar diffusion” strategy. The disc provides a dilution between the sample and the cells, and is, therefore, most suitable for solid yet soluble agents with high toxicity [6].

(3) For an extract elution experiment, the investigated sample is placed in a dish with cell culture medium for one or multiple days. During this time, toxins leach from the sample into the medium, after which the medium is added to the investigated cells. This strategy finds its main application in solid and multi-compound samples, which do not come into direct contact with cells or where the most prominent toxin(s) from all constituents is/are unknown [6]. However, it can also be used to study the long-distance communication between cell types, by adding medium conditioned by one cell type to the culture vessel containing the other cell type [7].

Like every cell experiment, cytotoxicity assays require appropriate controls [8]. Besides the negative control (untreated cells) and positive control (maximum cytotoxicity signal: adding an agent that lyses or kills all cells), for most assays, a background control sample without cells is required.


Cytotoxicity assays

Table 1 displays the most commonly used cytotoxicity assays and dyes, with the underlying mechanism and other basic properties indicated per experiment.

Five main cell properties – related to cell viability and behavior – are investigated in these assays [3, 6, 8]. Please note that absolute values related to these quantities can vary between cell types.

(1) Membrane integrity: healthy and viable cells have membranes impermeable to most molecules, whereas the membrane integrity for dead or damaged cells is compromised.

(2) Metabolic activity: damaged cells become less metabolically active.

(3) Cell contents: total amounts of DNA, proteins, ATP, or lysosomes can indicate the cell’s status and health.

(4) Cell proliferation: damaged cells reduce their proliferative activity.

(5) Cell attachment: damaged cells get released from their environment.


These cell properties are captured in the cytotoxicity experiments, via four underlying principles [1, 3, 9]:

(1) Dye binding: cell properties determine how much of the added dye binds to the investigated cells, after which excessive dye is washed away, and the signal of the bound dye is quantified and/or localized.

(2) Dye conversion: cell properties determine how much of the added dye is converted into a related molecule with deviant colorimetric or fluorometric properties, which is in turn quantified and/or localized.

(3) Dye uptake/exclusion: dyes can only enter and/or be retained in cells with specific properties.

(4) Cell imaging: cell properties are quantified from brightfield images.


Table 1 | Overview of cytotoxicity assays [1, 3-6, 8, 9]. *Addition of a dye can affect cell viability or behavior: live monitoring cytotoxicity experiments with a dye generally have a limited duration [8, 10, 11].

Assay/dyePrincipleMeasured cell propertyRead-out typeTime profileMechanism
Acridine orange (AO)dye bindingDNA contentfluorometriclive monitoring possible *membrane-permeable to all cells, and binding to DNA; used as counterstaining for e.g. PI, EtBr
Calcein acetoxymethyl ester (calcein-AM)dye conversionmetabolic activityfluorometriclive monitoring possible *membrane-permeable to all cells, converted into a fluorescent probe by metabolically active cells
Cell confluence quantificationimagingcell proliferationbrightfield imaginglive monitoringvitality-dependent cell growth and proliferation
Cell number quantificationimagingcell proliferationbrightfield imaginglive monitoringvitality-dependent cell proliferation
Cell size/shape quantificationimagingcell attachmentbrightfield imaginglive monitoringvitality-dependent cell attachment and morphology
Crystal violetdye bindingcell attachmentcolorimetricendpoint onlydye staining attached cells, assuming that detached cells are not viable
Ethidium bromide (EtBr)dye exclusion; dye bindingmembrane integrityfluorometriclive monitoring possible *membrane-impermeable to intact cells, binding to DNA of damaged cells
Ethidium homodimer (EthD)dye exclusion; dye bindingmembrane integrityfluorometriclive monitoring possible *membrane-impermeable to intact cells, binding to DNA of damaged cells
Glucose-6-phosphate dehydrogenase (G6PD) assaydye conversionmembrane integrityfluorometricrepeated medium samplingenzyme released from damaged cells, converting reporter dye in culture medium
Lactate dehydrogenase (LDH) assaydye conversionmembrane integritycolorimetricrepeated medium samplingenzyme released from damaged cells, converting reporter dye in culture medium
Neutral reddye uptakelysosome integritycolorimetricendpoint onlydye uptake and retention in lysosomes of viable cells, to maintain pH
Propidium iodide (PI)dye exclusion; dye bindingmembrane integrityfluorometriclive monitoring possible *membrane-impermeable to intact cells, binding to DNA of damaged cells
Resazurindye conversionmetabolic activityfluorometriclive monitoring possible *dye conversion by a mitochondrial respiratory chain in metabolically active cells
Sulforhodamine B (SRB)dye bindingprotein contentcolorimetricendpoint onlydye binding to amino acid residues of cellular proteins
Trypan bluedye exclusionmembrane integritycolorimetricendpoint onlymembrane-impermeable to intact cells, only staining damaged cells
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)dye bindingDNA integritycolorimetricendpoint onlydye binding to double-strand DNA breaks in dying cells
Water-soluble tetrazolium salts (WSTs)dye conversionmetabolic activitycolorimetricendpoint onlydye conversion by a mitochondrial respiratory chain in metabolically active cells
2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT)dye conversionmetabolic activitycolorimetricendpoint onlydye conversion by a mitochondrial respiratory chain in metabolically active cells
3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT)dye conversionmetabolic activitycolorimetricendpoint onlydye conversion by a mitochondrial respiratory chain in metabolically active cells
5-carboxyfluorescein diacetate, acetoxymethyl ester (CFDA-AM)dye conversionmetabolic activityfluorometriclive monitoring possible *membrane-permeable to all cells, converted into a fluorescent probe by metabolically active cells
5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazoly)-3-(4-sulfophenyl)tetrazolium (MTS)dye conversionmetabolic activitycolorimetricendpoint onlydye conversion by a mitochondrial respiratory chain in metabolically active cells
Apoptosis marker stainingsdye bindingapoptosis markersfluorometricendpoint onlyimmunostaining of apoptosis markers (e.g., caspases, annexin V, cytochrome c)
ATP-based assaysdye conversionATP contentcolorimetric/ fluorometric/ luminometricendpoint onlydye conversion depending on ATP concentration
Other viability dyes (brand-specific)dye exclusion; dye bindingmembrane integrityfluorometriclive monitoring possible *membrane-impermeable to intact cells, binding to DNA of damaged cells
Real-time viability assaysdye conversionmetabolic activityluminometriclive monitoring possible *viable cells convert pro-substrate into a substrate, which generates a luminescent signal with luciferase

Cytotoxicity assays: results

Read-outs

In the aforementioned assays, cytotoxicity is assessed from direct experiment results providing cell numbers, subdivided into a number of viable cells, dead cells, and/or dying cells [12]. Assays directly counting these cells are sometimes referred to as “viability assays” [12], and can be performed using the CytoSMART Exact FL to determine the viability of cells in suspension or the CytoSMART Lux3 FL for live monitoring of adherent cells. In this contradistinction, a property of “cytotoxicity assays” would be that the (viable) cell number is calculated from an indirect measure combined with normalization to a negative control sample [12]. This is mostly seen in the assays measuring metabolic activity and dye conversion assays.


Derived measures

The (viable) cell counts from cytotoxicity assays can be used to calculate agent-specific measures providing insight into the effect of the agent on the cells, enabling more direct comparison of various agents [6, 13]. For example, the half-maximal inhibitory concentration (IC50) and half-maximal effective concentration (EC50) are the concentrations required for inhibiting and stimulating half of the maximum biological function, respectively. Here, the biological function can be cell viability, metabolic activity, etc. Since these measures refer to concentrations, they are exclusively used for chemical agents. The selectivity index (SI; the IC50 of healthy cells divided by the IC50 of cancer cells) describes how selective cytotoxic activity of an agent is for cancer cells, and therefore finds its application in cancer and drug research. The no observed adverse effect level (NOAEL) is the highest exposure level at which there are no significant increases in the frequency or severity of adverse effects between the exposed population and its appropriate negative control (please refer to the section “Exposure of cells to the exogenous agent”). Many other derived measures exist, each with a main application in the more specialized research areas [13].


Cytotoxicity assays: pros and cons

As indicated before, the choice for the cytotoxicity assay to be executed depends on the combination of an exogenous agent, cell type, and research question. Each assay type has its pros and cons relating to the corresponding read-outs and results, leading to the most suitable assay for a particular investigation.

In dye conversion- and metabolic activity-related assays, many low-activity cells can result in the same quantitative read-out as few high-activity cells. Therefore, these assays provide insight into the total activity of a sample, which is converted to a viable cell number assuming a constant activity per cell. On the other hand, experiments providing only the cell number lack information regarding cell health and behavior – except “viable”, “damaged”, or “dead” in certain assays [3].

Destructive cytotoxicity methods (indicated with “endpoint only” in Table 1) can be experimentally challenging, because these assays require many more samples and resources to obtain a time profile. Besides that, a choice between determining cell activity and cell number has to be made, without the option to perform both consecutively on the same sample. On the other hand, endpoint measurements enable more direct quantification of the desired read-outs, providing higher accuracy [1, 8].

To obtain insight into both cell number and cell activity, live monitoring using brightfield imaging – which can be performed using the CytoSMART Lux2, Lux3 BR, or Omni – could be combined with (semi-)destructive endpoint measurements [8, 9, 12]. With this strategy, the best of all worlds can be combined into one experiment, with minimal extra effort for the researcher.


Concluding remarks

Cytotoxicity assays are performed for many purposes, from fundamental research and quality control to disease research and toxicology. Each purpose will require its specific experimental setup as well as desired read-outs and results. With so many experimental methods available to measure cytotoxicity, it is essential to choose the right methods for the research question. Methods involving live-cell imaging can easily provide valuable information about cell number or morphology over time and can be related to cell activity data of the same samples – which are possibly originating from (destructive) endpoint measurements. However, live-cell imaging can be performed without the need to prepare dedicated samples. Combining the aforementioned methods provides the most complete information – and the fairest comparison since the same samples are included in all read-outs – about cell health when exposed to a certain exogenous agent.

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