The Use of Precision-Cut Lung Slices for Studying Innate Immunity to Viral Infections.
The Use of Precision-Cut Lung Slices for Studying Innate Immunity to Viral Infections.
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DOI：
10.1002/cpz1.505
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发表时间：
2022-08
期刊：
影响因子：
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通讯作者：
Johansson, Cecilia
中科院分区：
文献类型：
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作者：
Michalaki, Christina;Dean, Charlotte;Johansson, Cecilia
精密切割肺切片（PCLS）是研究下呼吸道细胞的新型工具。由于PCLS保留了肺的完整性和结构，因此它们构成了用于研究下呼吸道细胞的稳健模型。使用PCLS进行成像以前已经记录;然而，其他应用和技术也可以应用于PCLS，以增加其使用，从而减少每个实验所需的动物数量。我们提出了一个详细的协议，从小鼠肺产生PCLS。我们表明，培养的PCLS保持活力，至少8天的培养，RNA可以从组织中分离，并可以进行流式细胞术从PCLS获得的细胞。此外，我们证明，细胞因子和趋化因子可以检测到暴露于病毒的PCLS的培养上清液。总体而言，这些方案扩大了PCLS的使用，特别是在感染研究中。版权所有© 2022作者。由Wiley Periodicals LLC出版。 基本方案1：基本方案3：从PCLS中分离RNA、cDNA转化和RT-qPCR基本方案4：从PCLS中染色细胞用于流式细胞术基本方案5：体内RSV施用和离体PCLS RSV暴露
Precision‐cut lung slices (PCLS) are a novel tool to study cells of the lower airways. As PCLS retain the integrity and architecture of the lung, they constitute a robust model for studying the cells of the lower respiratory tract. Use of PCLS for imaging has been previously documented; however, other applications and techniques can also be applied to PCLS to increase their use and therefore decrease the number of animals needed for each experiment. We present a detailed protocol for generating PCLS from the murine lung. We show that cultured PCLS remain viable up to at least 8 days of culture, that RNA can be isolated from the tissue, and that flow cytometry can be carried out on the cells obtained from the PCLS. Furthermore, we demonstrate that cytokines and chemokines can be detected in the culture supernatants of PCLS exposed to viruses. Overall, these protocols expand the use of PCLS, especially for infection studies. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Precision‐cut lung slices (PCLS) Basic Protocol 2: PCLS culture and viability Basic Protocol 3: RNA isolation from PCLS, cDNA conversion, and RT‐qPCR Basic Protocol 4: Staining of cells from PCLS for flow cytometry Basic Protocol 5: In vivo RSV administration and ex vivo PCLS RSV exposure