在过去的20年里,更大的蜡蛾的幼虫,梅洛内拉广场,作为用于研究人类病原体的体内哺乳动物替代模型生物,其普及程度迅速提高。对感染反应的实验读数通常仅限于观察黑化级联反应和量化幼虫死亡,而转录组学和蛋白质组学方法,还使用了确定微生物负荷的方法,随着时间的推移,更全面的分析感染的工具包可以改变该模型的适用性。作为无脊椎动物,Galleria具有先天性免疫系统,该系统由体液成分和先天性免疫细胞(称为血细胞)组成。尽管存在血细胞亚型的信息,关于其确切数量和功能的报告相互矛盾。流式细胞术先前已用于测定Galleria血细胞,但方案包括离心和固定-物理方法,这些方法有可能在分析前影响血细胞形态。这里,我们提出了一种通过流式细胞术分析活血细胞的方法,揭示Galleria血细胞只构成一个可解析的群体,基于相对大小或内部复杂性。使用荧光酵母聚糖颗粒,我们扩展了我们的方法,以表明高达80%的Galleria血细胞群显示吞噬能力。最后,我们证明了开发的检测方法可靠地复制了体外数据,表明白色念珠菌掩盖细胞壁的β-1,3-葡聚糖会破坏吞噬反应。因此,我们的方法提供了一种新的工具,可用于快速评估Galleria的吞噬作用和了解活体感染动态.
Over the last 20 years, the larva of the greater waxmoth, Galleria mellonella, has rapidly increased in popularity as an in vivo 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, Galleria 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 Galleria 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 Galleria 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 Galleria haemocyte population display phagocytic capability. Finally, we demonstrate that the developed assay reliably replicates in vitro data, showing that cell wall β-1,3-glucan masking by Candida albicans subverts phagocytic responses. As such, our method provides a new tool with which to rapidly assess phagocytosis and understand live infection dynamics in Galleria.