Flow cytometric distinction of early apoptotic, late apoptotic and primary necrotic morphological markers in Saccharomyces cerevisiae.

Regulated cell death (RCD) is a fundamental biological process that ensures tissue homeostasis, mediates stress responses, and contributes to diverse pathological conditions. The budding yeast Saccharomyces cerevisiae has emerged as a valuable model organism for the study of RCD, providing evolutionary insights into conserved molecular pathways and enabling the systematic dissection of cell death mechanisms in a genetically tractable system. Upon a lethal stimulus, yeast populations may segregate into three major subtypes: primary necrotic cells with disrupted plasma membranes, early apoptotic cells exhibiting morphological markers of apoptosis, and late apoptotic (or secondary necrotic) cells that display both apoptotic and necrotic features. Discriminating between these subpopulations is essential for accurate interpretation of RCD dynamics and has been facilitated by the transition from fluorescence microscopy to flow cytometry, which allows rapid, quantitative, and high-throughput analysis. This manuscript provides a methodological framework for the flow cytometric identification and quantification of these distinct subpopulations of dead or dying yeast, enabling reproducible and detailed assessment of cell death heterogeneity.