Medical Research Council Centre for Medical Mycology at the University of Aberdeen, Aberdeen Fungal Group, Institute of Medical Sciences, Foresterhill, Aberdeen, UK.
MRC-University of Glasgow Centre for Virus Research, Glasgow, UK.
Methods Mol Biol. 2021;2260:155-178. doi: 10.1007/978-1-0716-1182-1_11.
Phagocytosis and cytokine production are important processes by which innate immune cells, especially professional phagocytes such as neutrophils and macrophages, control and regulate immunity to fungi. These cellular responses are initiated when conserved pathogen components, known as pathogen-associated molecular patterns (PAMPs), are recognized by pattern-recognition receptors (PRRs), which include members of the C-type lectin receptor (CLR) family that are able to bind to fungal cell wall-derived carbohydrates. Phagocytosis and cytokine production can be quantitatively examined by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively, using in vitro based assays with primary-derived murine cells and cell lines. Here, we describe a flow cytometry-based method using transduced cell lines to assess the ability of CLRs to mediate internalization, using A. fumigatus conidia and the β-1,3 glucan receptor, Dectin-1 (CLEC7A), as an example. The use of ELISA-based assays to measure cytokine production by immune cells that are induced in response to fungi and methods for isolating and culturing primary macrophages from various murine tissues are described.
吞噬作用和细胞因子产生是先天免疫细胞（尤其是中性粒细胞和巨噬细胞等专业吞噬细胞）控制和调节对真菌免疫的重要过程。当保守的病原体成分（称为病原体相关分子模式，PAMPs）被模式识别受体（PRRs）识别时，会引发这些细胞反应，其中包括能够结合真菌细胞壁衍生碳水化合物的 C 型凝集素受体（CLR）家族的成员。可以通过流式细胞术和酶联免疫吸附测定（ELISA）分别定量检查吞噬作用和细胞因子产生，使用基于体外的原代衍生的鼠细胞和细胞系的测定法。在这里，我们描述了一种基于流式细胞术的方法，使用转导的细胞系来评估 CLR 介导内化的能力，以烟曲霉分生孢子和 β-1,3 葡聚糖受体 Dectin-1（CLEC7A）为例。还描述了用于测量针对真菌诱导的免疫细胞产生细胞因子的 ELISA 测定法以及从各种鼠组织中分离和培养原代巨噬细胞的方法。
