Measurement of bacterial capture and phagosome maturation of Kupffer cells by intravital microscopy.
Measurement of bacterial capture and phagosome maturation of Kupffer cells by intravital microscopy.
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DOI：
10.1016/j.ymeth.2017.05.004
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发表时间：
2017-09
期刊：
影响因子：
4.8
通讯作者：
Bas G J Surewaard;P. Kubes
中科院分区：
文献类型：
--
作者：
Bas G J Surewaard;P. Kubes
确定细菌是如何被吞噬细胞杀死的是细菌发病机理领域的核心。在吞噬过程中，微生物定位于吞噬溶酶体，在那里启动关键的防御机制，如酸化和活性氧（ROS）的产生。这一过程已经在体外进行了广泛的研究，然而，许多常驻组织吞噬细胞在从其自然环境中分离出来时会发生表型变化。因此，询问吞噬作用和吞噬体功能的细胞在其自然组织环境的背景下，增强了我们的理解的生物过程在体内。本文概述了一种实时活体显微镜协议，利用荧光染料来研究吞噬作用的过程，揭示了活动物宿主细胞内单个细菌的酸化和氧化。该技术的新奇存在于用荧光染料Oxyburst和pHrodo共价标记的细菌的使用中，所述荧光染料Oxyburst和pHrodo分别报告氧化或酸化。活体显微镜检查用于观察目标器官中报告细菌的摄取和随后的氧化或酸化。荧光标记的抗体可用于宿主免疫细胞如嗜中性粒细胞和巨噬细胞的复染，沿着参考染色剂以鉴定所有细菌。虽然这些试验最初是为了评估肝脏驻留巨噬细胞（枯否细胞）中金黄色葡萄球菌的摄取和存活而开发的，但该方案可适用于研究任何细菌-宿主细胞相互作用。
It is central to the field of bacterial pathogenesis to define how bacteria are killed by phagocytic cells. During phagocytosis, the microbe is localized to the phagolysosome where crucial defense mechanisms such as acidification and production of reactive oxygen species (ROS) are initiated. This process has extensively been studiedin vitro,however many resident tissue phagocytes will phenotypically change upon isolation from their natural environment. Therefore, interrogation of phagocytosis and phagosomal function of cells in the context of their natural tissue environment enhances our understanding of the biological processin vivo. This article outlines a real-time intravital microscopy protocol that utilizes fluorescent dyes to study the process of phagocytosis, which reveals acidification and oxidation of individual bacteria inside host cells of living animals. The novelty of this technique exists in use of bacteria that are covalently labelled with the fluorescent dyes Oxyburst and pHrodo, which respectively report on oxidation or acidification. Intravital microscopy is applied to visualize the uptake and subsequent oxidation or acidification of reporter bacteria in the organ of interest. Fluorescently labelled antibodies can be used to counter stain for host immune cells such as neutrophils and macrophages, along with reference stains to identify all bacteria. Although these assays were originally developed to assess the uptake and survival ofStaphylococcus aureusin liver resident macrophages (Kupffer cells), this protocol may be adapted to investigate any bacterium-host cell interaction.