Finding The Right Cell Proliferation/Viability Assays

Twenty years ago, researchers pretty much used trypan blue exclusion to test for cell viability, and 3H-thymidine incorporation to measure cell growth or proliferation, says Promega senior project manager Terry Riss. Yet while these tried-and-true assays are still done by many labs, there are far more options available today. Some techniques require state-of-the-art instrumentation and advanced training, while others can be used by anyone with basic laboratory hardware and the ability to follow a recipe. The choices of which to use depend not only how a lab is equipped, but on the sensitivity and throughput required, how many samples are to be assayed, how much information is needed from each sample, and a host of other considerations. Here are some.

For culturing, it’s important to know the density of the cells being passaged. The simplest way to do this is to remove a small aliquot, dilute it, and count the number of cells present with a hemacytometer or an automated cell counter. You can then extrapolate back to the number of cells in the sample. Yet because cultures – especially primary cell cultures -- contain dead cells, it’s also important to know how many cells in the aliquot are alive.

Viability markers measure the ability of a cell either to do something or fail to do something. Trypan blue, for example, is a measure of cell’s ability to exclude the dye. If the membrane is intact, cells are not stained. A similar dye -- which ATCC uses exclusively “because it’s more reliable” -- is erythrosine B, says Morgan, who oversees cell culture and cell biology kits for the repository. “The cells turn red when they are dead, like they turn blue with trypan. But it’s a lot clearer background, so you have fewer false negatives and false positives.”

These dyes are easy to use, and allow the researcher to examine the individual cells themselves, if so desired. But they are time-consuming and tedious to use for more than a few samples.

At least one other category of viability stains deserves mention, says Riss: nucleic acid binding dyes. Fluorescent molecules like propidium iodide (PI) and ethidium homodimer can enter membrane-damaged cells. When they intercalate with DNA they increases their fluorescent properties. Such reagents can be combined with those such as Calcein-AM that indicate metabolic activity, for what has become known as a “live/dead” assay. These are typically read by flow cytometry, but some have been adapted for microwells as well. “The disadvantage is that it requires a wash step -- there’s more manipulation involved,” notes Riss.

Many assays are now available – both as protocols and as kits – that utilize the ability of a viable cell to take up and effect a change in a probe, resulting in a change in absorbance, or fluorescence, or light emission after a sufficient incubation. Others generate a signal based ATP released from lysed cells.

Similarly, there are several colorimetric, fluorescent, and luminescent assays for viability that take advantage of the ability of enzymes in culture supernatant to cleave reporter substrates. Enzymes such as glucose 6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) are released by cells with damaged membranes, and are thus an indication of cell death. These tests have the advantage that they can be performed on aliquots of existing cultures.

These multi-well assays can be run in duplicate or triplicate, and thus can simultaneously interrogate the effects of many different parameters on cell viability. They are capable of automation, allowing for far more than 96 (or 384, or 1536) individual tests to be performed in a single run. Yet because they give a relative rather than absolute measure of the number of viable cells in a culture, results must be correlated to a standard curve. And because they look at populations rather than individual cells, no other information can be garnered from the assay.

There are reagents, however, that allow researchers to observe the proportion of viable (metabolically active) cells in real time. The redox indicator resazurin, for example, “does the same sort of thing as tetrazolium, except that you can watch in real time because it’s non-toxic. You just watch a color change,” says Morgan. “You can dump it into your cells, measure the color, and put the cells back, incubate it a bit more, take them out, measure the color. You can do it in real time during the process.”

Various forms of resazurin, such as dodecyl resazurin, can be used in cytometry. This vital dye can be combined with a variety of other markers in assays examining individual cells, to maximize the information gathered from a given experiment.