Bronietzki Marc, Kasmapour Bahram, Gutierrez Maximiliano Gabriel
Research Group Phagosome Biology, Helmholtz Centre for Infection Research.
Research Group Phagosome Biology, Helmholtz Centre for Infection Research; Division of Mycobacterial Research, National Institute for Medical Research;
J Vis Exp. 2014 Mar 10(85):51201. doi: 10.3791/51201.
Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosomal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.
吞噬细胞通过在其吞噬体中清除和消灭入侵的微生物，在先天免疫系统中发挥主要作用。吞噬体成熟是一个复杂且受到严格调控的过程，在此过程中，新生的吞噬体通过与细胞质中各种细胞器和区室精心编排的相互作用，经历剧烈的转变。这个过程对于吞噬细胞的生理功能至关重要，因为它赋予吞噬体溶解和杀菌特性，最终导致吞噬体与溶酶体融合以及吞噬溶酶体的生物发生，而这被认为是吞噬体成熟的最后关键阶段。在本报告中，我们描述了一种基于活细胞成像的方法，用于定性和定量分析溶酶体向吞噬体内容物递送的动态过程，这是吞噬溶酶体生物发生的一个标志。该方法使用包被免疫球蛋白G的微珠作为吞噬作用的模型，以及荧光团偶联的葡聚糖分子作为溶酶体腔内货物探针，以便通过延时成像和共聚焦激光扫描显微镜实时跟踪活巨噬细胞中溶酶体内容物向吞噬体的动态递送。在这里，我们详细描述了背景、制备步骤和逐步实验设置，以便在其他实验室轻松精确地应用此方法。我们所描述的方法简单、稳健，最重要的是，可以很容易地适用于研究不同系统以及各种实验条件下的吞噬体相互作用和成熟，例如使用各种吞噬细胞类型、功能丧失实验、不同探针和吞噬颗粒。
