Assessing anti-fungal activity of isolated alveolar macrophages by confocal microscopy.
Assessing anti-fungal activity of isolated alveolar macrophages by confocal microscopy.
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DOI：
10.3791/51678
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发表时间：
2014-07
期刊：
影响因子：
--
通讯作者：
Melissa J. Grimm;Anthony C. D’Auria;B. Segal
中科院分区：
文献类型：
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作者：
Melissa J. Grimm;Anthony C. D’Auria;B. Segal
肺是宿主细胞常规暴露于微生物和微生物产物的界面。肺泡巨噬细胞是遇到吸入真菌和其他微生物的第一线吞噬细胞。巨噬细胞和其他免疫细胞通过病原体识别受体识别曲霉基序并启动下游炎症反应。吞噬细胞NADPH氧化酶产生活性氧中间体（ROIs），对宿主防御至关重要。虽然NADPH氧化酶对中性粒细胞介导的宿主防御至关重要，但NADPH氧化酶在巨噬细胞中的重要性尚不明确。本研究的目的是描述巨噬细胞中NADPH氧化酶在介导宿主对烟曲霉的防御中的具体作用。我们发现肺泡巨噬细胞中的NADPH氧化酶控制被吞噬的烟曲霉孢子的生长4。在这里，我们描述了一种评估小鼠肺泡巨噬细胞（AMs）控制被吞噬的曲霉孢子（分生孢子）生长能力的方法。肺泡巨噬细胞在体内染色，10天后通过支气管肺泡灌洗（BAL）从小鼠中分离。将巨噬细胞镀在玻璃罩上，然后播种表达绿色荧光蛋白（GFP）的烟曲霉孢子。在特定时间，将细胞固定，并通过共聚焦显微镜评估带有被吞噬孢子的完整巨噬细胞的数量。
The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.