Characterising phagocytes and measuring phagocytosis from live Galleria mellonella larvae.

Over the last 20 years, the larva of the greater waxmoth, , has rapidly increased in popularity as an mammalian replacement model organism for the study of human pathogens. Experimental readouts of response to infection are most often limited to observing the melanization cascade and quantifying larval death and, whilst transcriptomic and proteomic approaches, and methods to determine microbial load are also used, a more comprehensive toolkit of profiling infection over time could transform the applicability of this model. As an invertebrate, harbour an innate immune system comprised of both humoral components and a repertoire of innate immune cells - termed haemocytes. Although information on subtypes of haemocytes exists, there are conflicting reports on their exact number and function. Flow cytometry has previously been used to assay haemocytes, but protocols include both centrifugation and fixation - physical methods which have the potential to affect haemocyte morphology prior to analysis. Here, we present a method for live haemocyte analysis by flow cytometry, revealing that haemocytes constitute only a single resolvable population, based on relative size or internal complexity. Using fluorescent zymosan particles, we extend our method to show that up to 80% of the haemocyte population display phagocytic capability. Finally, we demonstrate that the developed assay reliably replicates data, showing that cell wall β-1,3-glucan masking by subverts phagocytic responses. As such, our method provides a new tool with which to rapidly assess phagocytosis and understand live infection dynamics in .