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{"title":"Isolation, Purification, and Comprehensive Flow Cytometry Assessment of Lung Stromal Cells","authors":"Sophia Rottmann, Veronika Lukacs-Kornek","doi":"10.1002/cpz1.70078","DOIUrl":null,"url":null,"abstract":"<p>Stromal cells are non-hematopoietic cells that consist of endothelial cells and various mesenchymal cell populations. The composition of the stromal cell compartment is diverse in different organs. Numerous recent studies demonstrated that the lung environment contains heterogeneous mesenchymal stromal cell populations with distinctive genomic signatures and location preferences. Besides their role in supporting organ structure and remodeling tissue, mesenchymal stromal cells fulfill critical immune functions. These stromal cells show alterations during lung fibrosis and infectious disorders like COVID-19 or flu infection.</p><p>To date, their identification and isolation were challenging, and most information about their heterogeneity was derived from scRNAseq data. In this protocol, we describe an isolation, comprehensive flow cytometry assessment, and purification strategy for murine lung stromal cells. The described method is optimized for minimizing cell death while keeping a high level of cell purity. This protocol can be also used for ex-vivo analysis of these cells in downstream functional assays. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Isolation of stromal cells from murine lung tissue</p><p><b>Basic Protocol 2</b>: Flow cytometry assessment of lung stromal populations</p><p><b>Basic Protocol 3</b>: Purification of lung fibroblastic stromal cells</p><p><b>Alternate Protocol</b>: Positive selection of fibroblastic stromal cells</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70078","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current protocols","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpz1.70078","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
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Abstract
Stromal cells are non-hematopoietic cells that consist of endothelial cells and various mesenchymal cell populations. The composition of the stromal cell compartment is diverse in different organs. Numerous recent studies demonstrated that the lung environment contains heterogeneous mesenchymal stromal cell populations with distinctive genomic signatures and location preferences. Besides their role in supporting organ structure and remodeling tissue, mesenchymal stromal cells fulfill critical immune functions. These stromal cells show alterations during lung fibrosis and infectious disorders like COVID-19 or flu infection.
To date, their identification and isolation were challenging, and most information about their heterogeneity was derived from scRNAseq data. In this protocol, we describe an isolation, comprehensive flow cytometry assessment, and purification strategy for murine lung stromal cells. The described method is optimized for minimizing cell death while keeping a high level of cell purity. This protocol can be also used for ex-vivo analysis of these cells in downstream functional assays. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.
Basic Protocol 1 : Isolation of stromal cells from murine lung tissue
Basic Protocol 2 : Flow cytometry assessment of lung stromal populations
Basic Protocol 3 : Purification of lung fibroblastic stromal cells
Alternate Protocol : Positive selection of fibroblastic stromal cells
基质细胞是一种非造血细胞,由内皮细胞和各种间充质细胞群组成。不同器官的基质细胞组成各不相同。最近的大量研究表明,肺部环境中含有异质性间充质基质细胞群,它们具有独特的基因组特征和位置偏好。间质基质细胞除了在支持器官结构和重塑组织方面发挥作用外,还具有重要的免疫功能。这些基质细胞在肺纤维化和感染性疾病(如 COVID-19 或流感感染)期间会发生改变。迄今为止,间质基质细胞的鉴定和分离都很困难,有关其异质性的大部分信息都来自 scRNAseq 数据。在本方案中,我们介绍了小鼠肺基质细胞的分离、综合流式细胞仪评估和纯化策略。所描述的方法经过优化,可最大限度地减少细胞死亡,同时保持较高的细胞纯度。该方案还可用于在下游功能测试中对这些细胞进行体外分析。© 2024 作者。当前协议》由 Wiley Periodicals LLC 出版。基本方案 1:从小鼠肺组织中分离基质细胞 基本方案 2:流式细胞术评估肺基质群 基本方案 3:纯化肺成纤维基质细胞 替代方案:成纤维基质细胞的阳性选择。
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