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从小鼠肺中制备原代肺泡I型上皮细胞的实验方案。

Protocol for preparation of primary alveolar epithelial type I cells from mouse lungs.

作者信息

Huang Lulu, Tang Amy, Shao Bojing

机构信息

Laboratory of Vascular Inflammation and Thrombosis Research, Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065, USA.

Laboratory of Vascular Inflammation and Thrombosis Research, Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065, USA.

出版信息

STAR Protoc. 2024 Dec 20;5(4):103484. doi: 10.1016/j.xpro.2024.103484. Epub 2024 Dec 12.

DOI:10.1016/j.xpro.2024.103484
PMID:39671286
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11697548/
Abstract

Currently, the only approach for obtaining alveolar epithelial type I (AT1) cells is through the differentiation of cultured AT2 cells, which has several limitations. Here, we present a protocol to prepare AT1 cells directly from mouse lungs. We detail the steps for dissociating epithelial cells from the alveoli, followed by the purification of AT1 cells. Additionally, we describe how to assess the purity of AT1 cells using immunofluorescence staining and flow cytometry, along with approaches for verifying their functions.

摘要

目前，获取肺泡I型上皮细胞（AT1）的唯一方法是通过培养的AT2细胞分化，这存在一些局限性。在此，我们展示了一种直接从小鼠肺中制备AT1细胞的方案。我们详细介绍了从肺泡中解离上皮细胞的步骤，随后是AT1细胞的纯化。此外，我们描述了如何使用免疫荧光染色和流式细胞术评估AT1细胞的纯度，以及验证其功能的方法。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e2e/11697548/315b46ddecc7/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e2e/11697548/47dffda05782/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e2e/11697548/89de518d39de/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e2e/11697548/446f99980ed8/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e2e/11697548/315b46ddecc7/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e2e/11697548/47dffda05782/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e2e/11697548/89de518d39de/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e2e/11697548/446f99980ed8/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e2e/11697548/315b46ddecc7/gr3.jpg

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本文引用的文献

1
Isolation and culture of mouse alveolar type II cells to study type II to type I cell differentiation.分离和培养小鼠肺泡 II 型细胞以研究 II 型细胞向 I 型细胞的分化。
STAR Protoc. 2020 Dec 31;2(1):100241. doi: 10.1016/j.xpro.2020.100241. eCollection 2021 Mar 19.
2
Knockout Mice Reveal a Major Role for Alveolar Epithelial Type I Cells in Alveolar Fluid Clearance.基因敲除小鼠揭示了肺泡Ⅰ型上皮细胞在肺泡液体清除中的主要作用。
Am J Respir Cell Mol Biol. 2016 Sep;55(3):395-406. doi: 10.1165/rcmb.2016-0005OC.
3
Isolation and culture of alveolar epithelial Type I and Type II cells from rat lungs.
从大鼠肺中分离和培养I型和II型肺泡上皮细胞。
Methods Mol Biol. 2013;945:145-59. doi: 10.1007/978-1-62703-125-7_10.