High-throughput cell cycle and morphological analysis of Leishmania mexicana and other kinetoplastids

Analysis of the cell cycle in kinetoplastid parasites involves the assessment of the replication of single copy organelles, such as the nucleus, kinetoplast, and flagellum, alongside the observation of cell cycle stage-associated morphological changes, e.g., cell shape changes and the appearance of a mitotic spindle or cytokinesis furrow, which together allow the cell cycle stage of individual parasites to be determined. To date, most kinetoplastid cell cycle analysis has been performed using light microscopy and/or flow cytometry of fixed cells, but while these methods have proven highly valuable, microscopy can be time-consuming and flow cytometry can lack resolution. We have previously shown that imaging flow cytometry offers significant benefits for depth and speed of analysis. This is due to its ability to directly link the high-throughput and quantitative nature of standard flow cytometry with the visual and spatial data of microscopy, over an extensive array of morphological and fluorescence parameters, which can be calculated for both brightfield and fluorescence images of each cell. Furthermore, the ability to automate image analysis ensures high throughput. Here, we provide a step-by-step guide to analyzing the cell cycle of live promastigote Leishmania mexicana using imaging flow cytometry. We outline a method for quantitative DNA staining in live L. mexicana promastigotes using Vybrant™ DyeCycle™ Orange and provide protocols, guidance, and example analysis templates for using an ImageStream MkII imaging flow cytometer (Cytek) to acquire and analyze brightfield and fluorescence images of the parasite to determine cell cycle stage. We also detail how to employ mNeonGreen tagging of the orphan spindle kinesin, KINF, to provide greater resolution of cell cycle position. Our automated masking and gating pipeline enables rapid, high-throughput and semi-automated analysis of the L. mexicana cell cycle in live cells, in near real time, offering many advantages over conventional analysis methods. In addition, we envisage that this pipeline could be adapted to allow similar high-throughput analysis of the cell cycle of other kinetoplastid species and outline the approaches that could be taken to achieve this. [Abstract copyright: © 2026. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.]