Cellular assays are incredibly powerful and versatile research tools for measuring the functional activity of a target. An assay can help explore a cell’s biochemistry to understand how a genetic mutation behind a particular phenotype works, for instance. Live cells in the assays offer dynamic real-life models for biological outcomes, which is also very useful for drug discovery. Although there are three major types of assays—those that measure cell viability, cytotoxicity or apoptosis—choosing the optimal format is often daunting, especially when investigating multiple targets. With so many cell-based assays on the market, how do you select the best one for your research? Highlighted below are some considerations.
Identify meaningful endpoints
Most cell-based assays require more manipulation than biochemical assays and rely on specific endpoints. Choosing the best assay format starts with a thorough understanding of what endpoints will be measured and when (i.e., capturing relevant dosage and exposure periods). For instance, the total amount of caspase—a marker for apoptosis—remaining in the cells after exposure to tamoxifen is typically only a fraction of earlier time points after 24 hours, but toxicity varies widely throughout this duration. Exactly what assay endpoints to capture in relation to treatment, toxin exposure and test compound concentration are just some of the considerations for collecting meaningful data.
Choose the right cell type early
Choosing the right cell system early, based on the biology of the target, is key. Cells must be amenable to the assay, faithfully represent the system and express the necessary factors and signaling intermediates. Among the commonly used cells, fibroblasts (HEK293, Cos cells) are a conducive experimental system; however, they may not be the most relevant for your research. Cancer lines (HepG2 and PC-3 cells) may be a better choice and are generally easy to use, but remember, a cancer line contains mutations that can affect your experimental outcome. For instance, the cancer-cell line MCF-7 lacks a functional caspase-3 gene product, so if you are studying apoptosis using a DEVD substrate, you may underestimate it. Primary cells (HUVECs and hepatocytes) may produce the most accurate picture of the typical in vivo situation, but they can be difficult to grow and transfect.
Also, if you are performing a cell-viability assay that uses a metabolite (e.g., ATP) you need to remember that cell lines generally have a higher metabolic rate than primary cells. Primary cells in culture have a higher spontaneous rate of death, so if you are performing a cytotoxicity assay (e.g., LDH release), you can expect a higher background from an assay when using primary cells. The cell type will affect how you design your assay conditions.
Know your model system
A solid understanding of the cell-death process in your model system is important, too. For instance, some markers are only expressed transiently. During apoptosis, cells eventually stop metabolizing, lose membrane integrity and release cytoplasmic matter into the medium. Caspase activity is expressed only briefly and therefore is not the best indicator for signaling apoptosis as the primary mechanism of cell death. Cells in vitro fail to express apoptotic markers when undergoing rapid necrosis, as there isn’t sufficient time or cell energy to do so. Understanding the changes that correlate with cell death helps define endpoint selection, as well.
Comparison shop
All cellular assays require multiple steps, such as the removal of culture medium, cell washes and centrifugation. Cells can produce misleading results based on experimental variability in temperature, pH, media or serum concentration. To eliminate the potential for introducing user error, evaluate an assay’s ease of use. Compare the protocol requirements of similar assays before purchasing. For instance, some assays can generate a signal in minutes, offering an advantage over those requiring one to four hours of incubation. Rapid assays also reduce the possibility of artifacts created when test compounds interact with assay chemistry. Overall, you want an assay that’s simple to use but capable of providing a continuous record of cellular activity. Additional factors to consider include: reagent stabilities for signal strength, sensitivity of detection and multiplexing capabilities. (Note: You can improve detection sensitivity usually by increasing the incubation time, regardless of the assay.)
Summary
Cell-based assays differ from traditional enzyme- or antibody-based assays in that live cells require special considerations. The road to success begins with selecting the best cells for your research question and having a full understanding of meaningful endpoints to capture. Then, armed with your selected assay, you can proceed with full confidence.