Optimized MTT Assay for Metabolic Activity Evaluation in Human Spermatozoa

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is low-cost, widely available, and mainly used to assess cell viability as a function of cellular oxidoreductase enzyme-dependent conversion of the water-soluble tetrazolium salt to insoluble purple formazan. Although MTT is often associated with mitochondrial redox activity, extra-mitochondrial MTT reduction has been identified in several cell types, shaping the assay towards a total oxidoreductase/metabolic activity measurement. Standard MTT protocols were developed for adherent monolayer cells and perform poorly with suspension cells, as medium removal, cell and formazan loss, aggregate formation, and incomplete formazan solubilization result in data with high variability and nonlinearity. To overcome these limitations, here we describe an easy, inexpensive, and optimized MTT protocol for evaluating the metabolic activity in human spermatozoa. Key improvements in cell number optimization, compound and medium non-interference validation, post-assay cell preservation, and homogenous formazan solubilization resulted in a reproducible and linear protocol compatible with conventional microplate readers. This method quantifies spermatozoa metabolic activity with improved precision across technical replicates and can be directly applicable to toxicology screening, spermatozoa quality assessment, and fertility research focused on spermatozoa bioenergetics. Furthermore, although optimized for human spermatozoa, the workflow described in this protocol is readily adaptable to other suspension cell types following simple cell-specific calibration. © 2026 Wiley Periodicals LLC. Basic Protocol: Quantitative evaluation of human spermatozoa metabolic activity using MTT.