Live-cell kinetic (or Real time) assays show cellular dynamics that are often missed during experiments that use endpoint assays. Shown here is a method to measure the real-time exposure of phosphatidylserine (PS) on the outer leaflet of cell membranes during the apoptotic process.Credit: Promega
Although chimeric antigen receptor (CAR)-T therapy — where a patient’s T cells are engineered to fight cancer — works well against blood cancers in young people, that success drops dramatically for older patients. The impact of ageing on an older patient’s T cells, so-called cell fitness, could be the problem.1 “By measuring key markers of cell health and metabolic state it should be possible to determine whether CAR-T cells are developing correctly,” says James Cali, director of research for the assay design department at Promega, a biotechnology company in Madison, Wisconsin. “By providing assays for those markers, we hope to help scientists understand the fitness of CAR-T cells for their intended therapeutic purpose.”
For CAR-T cells and other applications, scientists would like to track the health of cells in real time. Most cell health assays are endpoint assays, often including a reagent that kills the cells. As a result, these assays provide just one reading, and tracking the time course of changes in cell health requires multiple experiments — say, one at 30 minutes after a treatment, another at 60 minutes, and so on. With a kinetic assay, cells stay alive, and scientists can continuously monitor their health over a single experiment, saving time and resources.2,3
When developing a cell-based manufacturing process, kinetic assays can also reveal “the often-invisible changes in the level of key metabolites and energy sources present in the culture medium, such as glucose, lactate, glutamine and glutamate,” says Terry Riss, a cell biology specialist and ambassador for Promega’s outreach activities. This could ultimately help optimize and refine the process.
But the utility of kinetic assays extends beyond understanding cell dynamics in manufacturing processes; they serve as valuable tools for studying cellular responses to external stimuli. This broader utility allows researchers to investigate the dose- and time-dependent effects of drugs on cell behaviour.4
Shining a light on cell dynamics
To further explore the versatility and effectiveness of kinetic assays, Riss and his colleagues conducted a multiplex experiment to assess the impact of panobinostat, a treatment used for multiple myeloma, on K562 leukaemia cells. Although researchers at Promega use both fluorescence and bioluminescence for readouts, most of their cell health assays use bioluminescence. “In general, bioluminescence has a very low background luminescent signal,” says Riss. “As a result, signal-to-background values are much higher compared to using fluorescence.” Plus, such sensitive detection “often enables miniaturization to 384- or 1,536-well formats, making the protocols more efficient,” he adds.
In the multiplex experiment Riss’s group used the RealTime-Glo MT Cell Viability Assay to measure live cells and the CellTox Green Cytotoxicity Assay to measure dead cells. “Both bioluminescent and fluorescent reagents can be added once at the beginning of the incubation period, then data recorded repeatedly from the same plate,” Riss explains. With higher doses of panobinostat, both assays showed an increase in dead cells. These kinetic assays also showed the real-time change in dead cells and helped provide insights into treatment mode of action and the mechanisms of toxicity.
“An endpoint assay only gives you a snapshot of what’s going on with cells at one point in time,” concludes Cali, “but a kinetic assay is like watching a movie.”