Immunofluorescence-Mediated Detection of Respiratory Virus Infections in Human Airway Epithelial Cultures.
Immunofluorescence-Mediated Detection of Respiratory Virus Infections in Human Airway Epithelial Cultures.
复制标题
免疫荧光介导的人类气道上皮培养物中呼吸道病毒感染的检测。
DOI：
10.1002/cpz1.453
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发表时间：
2022-06
期刊：
影响因子：
--
通讯作者：
Scull MA
中科院分区：
文献类型：
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作者：
Gagliardi TB;Iverson E;DeGrace EJ;Rosenberg BR;Scull MA
多种病毒病原体以气道上皮细胞为目标进行感染，其影响范围从轻微的上呼吸道症状到感染个体的死亡。在这些病原体中，有最近发现的和/或突发的病毒，它们有时不能感染常用的、永生化的细胞系，并且呼吸道中的感染表型仍然未知。在过去的几十年里，人类气道上皮细胞培养已经发展起来，并已被证明是一种有用的模型系统，用于培养难以生长的病毒，并在生理相关环境中分析感染的各种特征。本文介绍了在气液界面上培养分化良好的人气道上皮细胞的方法，这种细胞能在体内再现气管/支气管的粘膜上皮。我们进一步详细介绍了用呼吸道病毒（特别是鼻病毒、流感病毒和严重急性呼吸综合征冠状病毒2 (SARS - CoV - 2)）接种这些培养物的方法，并提供了一种通过免疫荧光显微镜检测双链RNA或病毒抗原阳性细胞的方案。这些技术，连同成像后分析，可用于表征感染效率和气道上皮内扩散动力学。此外，这些方法可以与针对细胞靶点的抗体结合使用，以确定感染期间细胞的趋向性和与特定宿主因子的共定位。©2022作者。Wiley期刊有限责任公司发表的当前方案：基本方案1：在空气-液体界面（HAE - ALI）培养人气道上皮细胞。基本方案2：病毒接种HAE - ALI。基本方案3：基于免疫荧光（IF）的检测HAE - ALI感染细胞
A diverse collection of viral pathogens target airway epithelial cells for infection, with effects ranging from mild upper respiratory tract symptoms to death of the infected individual. Among these pathogens are recently discovered and/or emergent viruses that sometimes fail to infect commonly used, immortalized cell lines and for which infection phenotypes in the respiratory tract remain unknown. Human airway epithelial cultures have been developed over the past several decades and have proven to be a useful model system in culturing hard‐to‐grow viruses and assaying various features of infection in a physiologically relevant setting. This article includes methods for the generation of well‐differentiated human airway epithelial cell cultures at air‐liquid interface that recapitulate the mucosal epithelium of the trachea/bronchus in vivo. We further detail inoculation of these cultures with respiratory viruses—specifically rhinovirus, influenza virus, and severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2)—and provide a protocol for the detection of double‐stranded RNA or viral antigen–positive cells by immunofluorescence microscopy. These techniques, together with a post‐imaging analysis, can be applied to characterize the efficiency of infection and kinetics of spread within the airway epithelium. Furthermore, these methods can be utilized in conjunction with antibodies against cellular targets to determine cell tropism and colocalization with specific host factors during infection. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of human airway epithelial cultures at air‐liquid interface (HAE‐ALI) Basic Protocol 2: Viral inoculation of HAE‐ALI Basic Protocol 3: Immunofluorescence (IF)‐based detection of infected cells in HAE‐ALI