How to Choose a Cell Viability or Cytotoxicity Assay

Choosing a cell health assay can be a challenging task. Here we provide a series of factors to consider when choosing cell-based assays for manual or automated systems.

What do you want to measure?

Which cells, viable or dead, do you want to detect at the end of an experiment? There are cell health assays available that specifically detect the number of living cells (viability assays), the number of dead cells (cytotoxicity assays) and for assessing the mechanism of cell death (apoptosis assays). If the information sought is simply to confirm whether there is a difference between “no treatment” negative controls and “toxin treatment” of experimental wells, the choice between measuring the number of viable cells or the number of dead cells may be irrelevant. However, if more detailed information on the mechanism of cell death is being sought, the duration of exposure to toxin, the concentration of the test compound, and the choice of the assay endpoint become critical (1). Explore the following considerations to help you determine what you want to measure and which assays can help you.

What is your model system?

The species of origin and cell types used in cell health studies are often dictated by specific project goals or the drug target that is being investigated. Regardless of the model system chosen, establishing a consistent and reproducible procedure for culturing cells and setting up assay plates is important. Variation in the number of cells per well or equilibration period prior to performing the assay may affect cellular physiology. Maintenance and handling of stock cell cultures at each step of the process should be standardized and validated for consistency. In addition, be sure to use known positive and negative controls throughout the course of the experiment to establish a general understanding of the physiological condition of the cells. Because the nature of the sample can vary depending on cell type and whether it is a 2D or 3D culture model, the assay procedure should be validated for each culture model system. A model system that requires use of smaller cell numbers, such as with primary cells or other limited cell types, will require an assay with increased sensitivity.

Are you using 2D or 3D cell cultures?

Cells in culture are only a model system and are different than cells in their normal in vivo environment. Many researchers are now using 3D cell cultures to more closely mimic in vivo conditions. Instead of growing in a monolayer on a plate surface, cells in 3D culture grow within a conformation that allows them to interact with each other, forming cell:cell connections. This added complexity can present challenges for experimental design when performing cell based assays because assay reagents may have difficulty reaching the center of large microtissues, and lytic assays may not be able to disrupt all cells within the 3D system. Assays may need to be optimized for 3D systems, by reformulating reagents with stronger detergents and incorporating mechanical disruption and longer incubation times. Colorimetric assays, such as MTT, are not optimal for use with 3D cell cultures because they have limited ability to penetrate multiple layers of cells.

The CellTiter-Glo® 3D Cell Viability Assay (Cat.# G9681) is specifically designed for determining cell viability in 3D culture models. The assay reagent has increased lytic capacity—allowing better penetration of large spheroid samples resulting in more accurate determination of viability compared to other assay methods. The CellTiter-Glo® 3D Assay Reagent measures ATP as an indicator of viability and generates a luminescent readout that is much more sensitive than colorimetric or fluorescence-based methods.

To measure cytotoxicity of a compound, the LDH-Glo™ Cytotoxicity Assay (Cat.# J2380) is ideal for use with 3D culture models. Instead of relying on penetration of the cell membrane, the assay measures LDH release from dead cells into the medium. The assay requires only small volumes of medium (2–5µl) to be removed, allowing for repeated sampling over time. This conserves microtissues and maintains remaining viable cells, allowing you to use samples for additional downstream applications, with other assays, or for nucleic acid analysis to acquire more data using the same samples.

When is the right time to perform a cell health assay?

Matching the detected assay marker to the information you need is vital to choosing the appropriate cell health assay. A basic understanding of the changes that occur during different mechanisms of cell death will help you decide which assay to choose. Figure 9 shows a simplified example illustrating chronological changes occurring during apoptosis and necrosis and the results that would be expected using assays that measure different markers.