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Resazurin Reduction-Based Assays Revisited: Guidelines for Accurate Reporting of Relative Differences on Metabolic Status
Open accessAbstract
Cell viability and metabolic activity are ubiquitous parameters used in biochemistry, molecular biology, and biotechnological studies. Virtually all toxicology and pharmacological projects include at some point the evaluation of cell viability and/or metabolic activity. Among the methods used to address cell metabolic activity, resazurin reduction is probably the most common. At variance with resazurin, resorufin is intrinsically fluorescent, which simplifies its detection. Resazurin conversion to resorufin in the presence of cells is used as a reporter of metabolic activity of cells and can be detected by a simple fluorometric assay. UV–Vis absorbance is an alternative technique but is not as sensitive. In contrast to its wide empirical “black box” use, the chemical and cell biology fundamentals of the resazurin assay are underexplored. Resorufin is further converted to other species, which jeopardizes the linearity of the assays, and the interference of extracellular processes has to be accounted for when quantitative bioassays are aimed at. In this work, we revisit the fundamentals of metabolic activity assays based on the reduction of resazurin. Deviation to linearity both in calibration and kinetics, as well as the existence of competing reactions for resazurin and resorufin and their impact on the outcome of the assay, are addressed. In brief, fluorometric ratio assays using low resazurin concentrations obtained from data collected at short time intervals are proposed to ensure reliable conclusions.
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![Figure 1 Fitting curves of the data presented by Uzarski et al. [7] on the reducing power of three different cell lines (panel A-TK188 fibroblasts, grey; Distal MDCK cells, blue; Proximal RPTE cells, orange) and ratio of the fitted curves obtained with any two out of the three cell lines (panel B-MDCK/RPTE, blue; MDCK/TK188, orange; TK188/RPTE, grey). For the fitted curves in panel A, k2/k1 was 0.11, 0.20 and 0.00 for MDCK, RPTE and TK188, respectively. [A](t = 0) was set equal for the three cell lines. Aditional data can be found in reference [7].](/paper-figures/1537ac7d59a543f5a904aa42eba3072d/resazurin-reduction-based-assays-revisited-guidelines-for-accurate-reporting-of-figure-1.jpg)
![Figure 2 Fitting curves of the data presented by Lavogina et al. [8] on the metabolic activity of HeLa cells in the absence (blue) and presence of 10 μM doxorubicin (orange) (panel A) and ratio of the two fitted curves (panel B). For the fitted curves in (panel A), k2/k1 was 14.4 and 0.00 for the sample with and without doxorubicin, respectively. [A](t = 0) was set equal in both conditions.](/paper-figures/cf69fcab79844a64b7591b1e0265a129/resazurin-reduction-based-assays-revisited-guidelines-for-accurate-reporting-of-figure-2.jpg)
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“Many commonly used in vitro viability assays, including tetrazolium-and resazurinbased assays, rely on cellular metabolic or redox activity as indirect indicators of viability instead of directly quantifying viable cell number or cell death (Ghasemi et al. 2023;Vieira-da-Silva and Castanho 2023).”
From Primary cell cultures versus immortalized cell lines: critical comparative insights into biological fidelity and translational applications (2026) · cites this paper as Vieira-da-Silva and Castanho 2023
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Principles of Fluorescence Spectroscopy
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Njoku, D. I., Guo, Q., Dai, W., Chen, J. L., Mao, G., Sun, Q., Sun, H., & Peng, Y. (2023) · TrAC Trends in Analytical Chemistry · doi:10.1016/j.trac.2023.117288 · Cited by 35
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Metadata
- Authors
- Vieira-da-Silva, B., & Castanho, M. A. R. B.
- Affiliations
- Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal
- University of Lisbon (PT)
- Corresponding author
- Castanho, Miguel A. R. B.
- Venue
- Molecules
- Publisher
- MDPI AG
- Published
- 2023-03-01
- Volume
- 28
- Issue
- 5
- Pages
- 2283
- ISSN
- 1420-3049 (ISSN-L)
- PMC
- PMC10005198
- OpenAlex
- W4322743436
- Cited by
- 76
- References
- 8
- Citations by year
- 2026: 34 · 2025: 24 · 2024: 12 · 2023: 6
- FWCI
- 6.77
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Molecules (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Portuguese National Funding Agency for Science, Research and Technology (RCAAP Project by FCT) (submittedVersion, other-oa)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- “la Caixa” Foundation and FCT (LCF/PR/HR21/00605)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › bioluminescence and chemiluminescence research
- Health Sciences › Veterinary › Small Animals › Animal testing and alternatives
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cancer and biochemical research
- Keywords
- resazurin reduction, resorufin, cell metabolic activity, cell viability, fluorometric assay, assay linearity, calibration, kinetic analysis, competitive reactions, assay interference
- MeSH terms
- Fluorometry, Indicators and Reagents, Oxazines, Xanthenes
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- Vieira-da-Silva, B., & Castanho, M. A. R. B. (2023). Resazurin Reduction-Based Assays Revisited: Guidelines for Accurate Reporting of Relative Differences on Metabolic Status. Molecules. https://doi.org/10.3390/molecules28052283
