A Universal Resazurin-Based Viability Assay for Prokaryotic and Eukaryotic Cells in 2D and 3D Cultures

In vitro cytotoxicity assessments frequently rely on staining-based methods that indirectly estimate viable cell numbers. A major limitation of many such techniques is their endpoint nature, requiring cell lysis or irreversible processing that precludes longitudinal monitoring of cellular responses following treatment. An ideal assay for evaluating cell viability and proliferation should be simple, rapid, cost-effective, reproducible, and highly sensitive, while also enabling accurate quantification with minimal interference from test compounds. The resazurin reduction assay satisfies these criteria, offering a sensitive and economical alternative to conventional tetrazolium-based methods. Although both assay types depend on the metabolic reduction of a dye by viable cells, they differ mechanistically. Tetrazolium salts (e.g., MTT) are reduced by cellular dehydrogenases to insoluble formazan crystals that require solubilization before detection. In contrast, resazurin-a cell-permeable, non-fluorescent blue dye-is reduced to resorufin, a highly fluorescent compound detectable without additional processing steps. This property renders the resazurin assay broadly applicable to viability testing in eukaryotic cells cultured in both 2D and 3D formats, as well as in bacterial systems. Here, we present a resazurin-based reduction assay across diverse experimental models, emphasizing its practicality, reproducibility, and adaptability for real-time viability monitoring. Key features • Realtime, nondestructive monitoring: Allows repeated measurements of the same samples over time without toxicity or disruption. • Simple "add-incubate-read" workflow: No cell lysis, washing, or extraction steps, reducing time and variability. • Broad sample compatibility: Works with 2D monolayers, 3D spheroids, and bacterial cultures. • High sensitivity and low background: Fluorescent detection of resorufin enables accurate quantification of small viable cell populations.