Choosing the Right Cell Health Assay

Cell-based models are critical for analyzing the relationships between test article exposures and subsequent biological responses. However, any experimental exposure using live cells may impact the overall health of the cell population, potentially complicating the interpretation of results. Cell health can be assessed from multiple angles, using a variety of assay options. This article explores the relevance and common methods of measuring these endpoints.

Live-cell viability

Viability assays measure surrogates of cellular vitality proportional to cell number, allowing differentiation between normal proliferative and induced non-proliferative states. These measures have exceptional utility for ascertaining the general health status of tested cells following an experimental exposure.

Viability is commonly established by assessing bioenergetic status. Tetrazolium dyes (MTT, XTT, WST-1) and resazurin-based probes are cost-effective, redox-sensitive compounds reduced by active cellular dehydrogenases into colorimetric or fluorescent products. These formats demonstrate strong sensitivity but require 4–8 hours of probe co-incubation. Bioluminescent assays obviate this requirement by introducing the probe at the beginning of the experimental exposure and allowing for continuous real-time measures.

ATP is a well-regarded viability surrogate because it is tightly regulated within cells, and extracellular ATP from dead cells is rapidly degraded by endogenous ATPases. Bioluminescent ATP assays are often considered the gold standard for viability methods for their sensitivity, simplicity, and technical reliability. These homogenous assay formats require less than five minutes of reagent contact time and offer significant improvements in throughput and scalability.

Unfortunately, all viability assay methods are subject to some degree of assay interferences, particularly from bioenergetic modulators developed for oncology applications. Orthogonal methods utilizing proteolytic biomarker activity provide non-destructive alternatives for cell number normalization and multiplexing.

It is important to note that viability assays cannot discriminate whether a reduced cell count is the result of arrested or stressed cells, or if they have progressed to necrosis or programmed cell death. Other assays must be used to reveal this specific information.

Proliferation

While viability assays assume that signal is proportional to cell number, certain experimental conditions can break that assumption. For example, standard viability chemistries are not well-suited to studies examining immune cell responsiveness or targeted perturbation of the cell cycle. Therefore, to unambiguously assess in vitro proliferative responses, researchers measure parameters that are directly associated with cell division. There are two main approaches for achieving this.

Incorporation assays introduce a non-natural, synthetic thymidine analog into cell cultures, which can be incorporated into newly synthesized DNA during the S-phase of replication. Although the classic radiometric format (³H-thymidine) provides the greatest sensitivity, it has been largely supplanted in high-throughput applications by colorimetric, fluorometric, and chemiluminescent detection formats (BrdU and EdU), which produce less dangerous and costly waste streams.

Protein biomarkers such as Ki-67 offer another means of assessing cell division. This nuclear antigen is expressed during all phases of actively cycling cells but is notably absent in non-proliferating populations. Various commercial endpoint immunodetection formats are available.

Cytotoxicity

If test conditions are intolerable, cells will lose membrane integrity and die. Cytotoxicity assays provide a robust means of quantifying the extent of this terminal phenotype within a cell population. Unlike viability assays, membrane integrity methods can positively discriminate between quiescent cells and necrotic phenotypes. These methods rely on either dead cells passively releasing enzymatic biomarkers into the extracellular environment or allowing unrestricted inward cell permeability of normally excluded probes.

Enzymatic release assays detect enzymes that leak from membrane-compromised cells. Lactate dehydrogenase (LDH) is the most favored biomarker because it is abundant, enzymatically active, and extracellularly stable. LDH can be measured using fluorescence or luminescence in homogeneous or supernatant sampling formats, delivering high dead cell sensitivity across commonly utilized cell models.

Non-LDH enzyme release methods provide additional options, including assays measuring proteolytic activity released after membrane integrity loss. Although proteolytic markers are less persistent than LDH, they offer advantages for multiplexing with viability measures or sequential caspase activation assays.

Enzymatic release approaches are particularly valuable when kinetic measurements are needed or when supernatant sampling is preferred to avoid interfering with live cells. On the other hand, all enzymatic activity is transient, so membrane-impermeant dyes offer an alternative for capturing the culmination of all cells that lost membrane integrity during the exposure period. Trypan blue, a common method for measuring cytotoxicity, binds to intracellular proteins when membranes are compromised. Propidium iodide and asymmetric cyanines bind to exposed DNA.

The choice between enzymatic and dye-based methods ultimately depends on whether temporal resolution of cell death events or cumulative detection of all membrane integrity loss is more relevant to the experimental question.

Dissecting programmed cell death

Understanding exactly how cells die helps researchers interpret experimental results and determine their next steps. While there are many types of programmed cell death, distinguishing between apoptosis (organized cell death) and necrosis (uncontrolled cell death) provides actionable insights about a compound’s mechanism or cellular stress response.

Apoptosis involves activation of caspase enzymes, particularly caspases-3 and -7, which serve as reliable markers of committed apoptotic cell death. These enzymes can be measured using fluorescent or bioluminescent detection methods. Bioluminescent formats offer higher sensitivity and can be multiplexed with viability and cytotoxicity assays in the same experiment.

Apoptotic cells also undergo active membrane remodeling, which exposes phosphatidyl serine (PS) to the outer membrane surface. PS can be measured using Annexin V-labeled probes in various detection formats, including real-time bioluminescent methods that can track PS exposure over 72 hours. Since dying cells lose membrane integrity regardless of death pathway, combining PS detection with membrane-impermeant DNA dyes helps distinguish early apoptosis from rapid necrotic death.

Conclusions

The first step to selecting a cell health assay is to determine the most relevant cell health measurement for your system and research goals. From there, each method can be assessed for cost, sensitivity, scalability, and ease-of-use. Ultimately, the best assay is the one that provides the most robust answer to the exact question you have in your research project.

Andrew L. Niles is a Senior Research Scientist in Promega’s Advanced Technology Group. His scientific focus is directed at providing innovative solutions for improving in vitro gene editing efficiencies allowing for integration of bioluminescent tags and reporters. His work seeks to explore and detail how these approaches and methods are uniquely enabling for studying endogenous cell biology during drug target validation and discovery activities. His past research efforts have led to multiple “add-mix-measure” and real-time assay systems for evaluating cell health and/or assigning mechanism of cytotoxicity. He has authored several well-cited publications and reviews and has been granted 14 U.S. Patents. Promega Corporation is a global leader in providing innovative solutions and technical support to the life sciences industry, offering more than 4,000 products used in research, diagnostics, forensics and applied testing worldwide.