Flow Cytometry Analysis of Fungal Ploidy

Ploidy, the number of sets of homologous chromosomes in a cell, can alter cellular physiology, gene regulation, and spectrum of acquired mutations. Advances in single cell flow cytometry have greatly improved our understanding of how genome size contributes to diverse biological processes including speciation, adaptation, pathogenesis and tumorigenesis. For example, fungal pathogens can undergo whole genome duplications during infection of the human host and during acquisition of antifungal drug resistance. However, quantification of ploidy is dramatically affected by the nucleic acid staining technique and the flow cytometry analysis of single cells. Ploidy in fungi is also impacted by samples that are heterogeneous for both ploidy and morphology, and control strains with known ploidy must be included in every flow cytometry experiment. To detect ploidy changes within fungal strains, we have developed the following protocol to accurately and faithfully interrogate single cell ploidy.