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 indirectly. 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 to 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 streamlined, universal protocol for implementing the resazurin reduction assay across diverse experimental models, emphasizing its practicality, reproducibility, and adaptability for real-time viability monitoring. Key featuresO_LIReal-time, non-destructive monitoring: Enables longitudinal studies by allowing repeated measurements of the same samples over hours without toxicity or disruption. C_LIO_LIStreamlined workflow: A simple "add-incubate-read" protocol eliminates the need for cell lysis, washing, or extraction, saving time and reducing variability. C_LIO_LIBroad sample compatibility: Versatile and reliable for use with 2D monolayers, 3D spheroids, organoids, and bacterial cultures. C_LIO_LIHigh sensitivity: Fluorescent detection of resorufin provides exceptional sensitivity, enabling accurate quantification of even small viable cell populations. C_LIO_LILow background and minimal interference: A clean fluorescent readout reduces the risk of signal artifacts, offering a more reliable alternative to traditional colorimetric assays. C_LIO_LICost-effective and accessible: Utilizes standard laboratory plate readers and commercially available reagents, making it an economical choice for any lab. C_LIO_LIScalable for high-throughput screening: Easily adaptable to various plate formats, supporting both small-scale experiments and large-scale automated screening applications. C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/718248v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@82bcecorg.highwire.dtl.DTLVardef@14164aforg.highwire.dtl.DTLVardef@395118org.highwire.dtl.DTLVardef@fb1349_HPS_FORMAT_FIGEXP M_FIG C_FIG