Sokolovska Anna, Becker Christine E, Stuart Lynda M
Developmental Immunology/CCIB Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts.
The Broad Institute of MIT and Harvard, Cambridge, Massachusetts.
Curr Protoc Immunol. 2012 Nov;Chapter 14:14.30.1-14.30.12. doi: 10.1002/0471142735.im1430s99.
Phagocytes are an important part of host defense, playing a critical role in innate immune responses against pathogens and in the initiation of adaptive immunity. One of the main characteristics of these cells is their ability to recognize and internalize invading microorganisms into a phagosome. The internalized microbe is rapidly delivered into a mature phagolysosome where it is killed and degraded. However, numerous pathogens have evolved complex mechanisms to manipulate these intracellular organelles to establish a survival niche. Here, we describe several methods to assess important properties of phagosomes in macrophages, such as phagocytosis, acidification of the phagosome contents during the maturation process, and the ability of phagosomes to inactivate and kill pathogens. Phagocytosis and phagosome acidification assays are FACS-based assays where labeled bacteria are used as probes to monitor internalization into a phagosome and to detect the pH of the phagosome environment. The killing assay is based on the counting of bacterial colonies after recovery of internalized bacteria from macrophages.
吞噬细胞是宿主防御的重要组成部分，在针对病原体的固有免疫反应以及适应性免疫的启动中发挥关键作用。这些细胞的主要特征之一是它们能够识别入侵的微生物并将其内化到吞噬体中。内化的微生物会迅速被递送到成熟的吞噬溶酶体中，在那里被杀死并降解。然而，许多病原体已经进化出复杂的机制来操纵这些细胞内细胞器，以建立一个生存小生境。在这里，我们描述了几种评估巨噬细胞中吞噬体重要特性的方法，例如吞噬作用、成熟过程中吞噬体内容物的酸化以及吞噬体使病原体失活和杀死病原体的能力。吞噬作用和吞噬体酸化测定是基于流式细胞术的测定，其中标记的细菌用作探针来监测内化到吞噬体中并检测吞噬体环境的pH值。杀伤测定基于从巨噬细胞中回收内化细菌后对细菌菌落的计数。
