Cell based assays are a mainstay of drug discovery and development. Yet with so many different types of cell-based assays available, how do you choose the right one for your experimental needs? This article looks at what some of the most prevalent of those assays are, how they relate to one another, and how to choose among them.

Why cell-based?

Using a cell-based assay allows us to interrogate the effects of compounds or genetic or environmental changes on more than just gene expression or protein secretion. It enables observation at a whole cell level of more complex events such as cell division, apoptosis, or cell death, explains Oksana Sirenko, Senior Manager of the Applications Team at Molecular Devices.

Assays can be performed on (or with) normal primary cells or immortalized cell lines, edited or transfected cells, patient-derived or stem cell-derived cells. They can be conducted on 2D or 3D cultures or single-cell suspensions. Generally speaking, the unifying factor is that the assays are performed in vitro on (typically) intact, live cells rather than a lysate or extraction. They look for functional changes, although these may be measured through biochemical means.

It’s alive (or is it?)

The most common cell-based assay is simply to count the number of live cells present. “The primary goal is often just to measure cell viability, but for almost any other cell-based assay it’s helpful to know how many cells are there at the end of the experiment,” says Terry Riss, Promega’s Scientific Ambassador, Cell Health. “You may want to know how many live cells are there, or how many dead cells are there. Or if they died, how did they die—was it an apoptotic event? All those pieces of information are key to understanding other aspects of cell-based assays.”

The classic definition of viability is based on membrane integrity. When a cell dies eventually its membrane begins to leak and things that were contained within the cell can escape into the cell culture medium—the cytosolic enzyme lactate dehydrogenase (LDH), for example. By adding a substrate that is converted by LDH into a quantifiable product such as fluorescence or luminescence, the number of non-viable cells in a heterogenous population can be estimated.

At the same time, things like dyes or substrates in the medium that were excluded from the live cell can now get into cells with compromised membrane integrity. Entry of so-called “vital dyes” such as propidium iodide and trypan blue into cells, for example, indicate non-viable cells.

A host of assays are based on the fact that metabolic activity diminishes and then ceases as cells die. Reagents such as a tetrazolium (MTT) can readily enter even live cells. The endogenous cellular machinery of viable cells will reduce the compounds, either changing their absorbance at a given wavelength (which can be measured by a colorimeter such as a standard plate reader), rendering them fluorescent (readable by a fluorometer), or both. These, says Riss, are probably “the most commonly used method to measure viability” outside of high-throughput screening labs.

In high-throughput screening labs, the most common is the CellTiter-Glo® (CTG) ATP assay. “As soon as a cell died it loses the ability to synthesize ATP, and the ATP that’s present is used up by all of the enzymes that use ATP—so ATP is used as a marker of viable cell number,” Riss explains. CTG lyses the cells and uses the ATP to allow the assay’s luciferin/luciferase reaction to produce luminescence. It’s the fastest and the most sensitive assay, with the least amount of things that interfere with it, he says. But the fact that it kills the cells limits what can be done with those cells afterward.

Multiplexing, in order

Oftentimes a researcher needs to know more than just what proportion of cells have survived, or thrived, after a treatment. Does that treatment cause the cells to proliferate, necrose, or undergo apoptosis, for example? Does it cause the cells to activate or inhibit a particular signaling pathway, or up- or down-regulate a certain receptor?

Many of the assays for these sequelae are performed on cells in culture (as opposed to lysed) cells. For example, annexin V can be added at the beginning of culture, along with propidium iodide, to identify apoptosing and necrosing cells, respectively. Homogenous versions of these assays allow real-time tracking without intervening wash steps. And while some assays can be performed together, or sequentially, it is important to know the chemistries involved—including the readouts, such as fluorescent excitation and emission wavelengths—to assure that they are compatible.

Cell lines are also available, or can be engineered, to query a specific event. “For example, if you wanted to set up a cell-based assay to measure infectivity of SARS coronavirus, or look at variants of the coronavirus, you could create a cell-based assay using a specific target cell either with or without the receptor,” explains Dzung Nguyen, Senior Director of Marketing at BPS Biosciences. “Then you would use something like a lentivirus that expresses the spike protein along with some sort of reporter that it delivers to the cell. Once infection happens you get a readout using something like luciferase or GFP.”

He cites another illustration of a cell-based assay using engineered cells, utilizing a cell line with a promoter sequence driving a luciferase reporter. A signaling pathway or receptor binding might activate a transcription factor that activates that promoter to induce expression of luciferase.

Imag(ine) that in 3D

Not all cell-based assays use just color, luminescence, or fluorescence as their readouts. “Imaging allows analysis of a wide variety of processes from cell death, proliferation, and apoptosis, to the appearance of differentiation markers and kinetic monitoring of calcium isolation,” Sirenko notes. “You can visualize changes in morphology, or complex processes like the contraction of cardiac cells.”

She sees an industry trend toward more complex cell-based assays, perhaps using phenotypic assays with high-content imaging, “or even 3D cell-based assays, for high-throughput screening.” 3D models represent some aspect of tissue functionality, like metabolic activity of the liver, or spikes in neuronal activity, or cell penetration, and more closely mimic the interactions between cell types.

How to choose

The first question Nguyen would ask is, “Is the cell system appropriate to accurately measure what you’re trying to measure?”

Riss asks you to consider what you want to know at the end. An experiment that simply counts live (or dead) cells may not directly measure proliferation or cytotoxicity, for example, or detail a drug’s mechanism of action.

Other questions to ask include: Do you want to multiplex assays—which costs more but is faster—or conduct the second assay on only a select few cells? Will the assays interfere with one another?

What is the readout? Imaging typically requires more complex setup, equipment, and analysis, but can yield correspondingly more information. Or do you prefer the sensitivity and speed of luminescence, the localization and multiplexing ability of fluorescence, or the frugality of colorimetry?

Is it important to minimize the steps, opting for homogenous assays?

For even more considerations on planning for and running cell-based assays, see “In Vitro Cell Based Assays,” part of the National Library of Medicine’s Assay Guidance Manual.

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