Multicompartmental analysis of the murine pulmonary immune response by spectral flow cytometry.
Multicompartmental analysis of the murine pulmonary immune response by spectral flow cytometry.
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DOI：
10.1152/ajplung.00317.2022
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发表时间：
2023-10-01
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影响因子：
--
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中科院分区：
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由于肺部的复杂结构和组成，肺部炎症的研究需要独特的考虑。肺部有多个隔室和不同的免疫细胞群，具有固有的高自身荧光，并参与宿主对肺部病原体的反应。我们描述了一种方案，通过一种新的方法组合来解释这些因素-体内区室分析和具有广泛抗体面板的光谱流式细胞术。体内区室分析可以精确定位边缘血管、肺间质、肺不可洗气道和肺可洗气道以及肺淋巴结内的免疫细胞。具有广泛抗体面板的光谱流式细胞术支持一种无偏倚的探索性方法来研究肺部炎症期间不同的免疫细胞群。最重要的是，光谱流使用细胞自身荧光来帮助免疫细胞群的分辨和鉴定。这种方法能够获得与知情门控和降维算法兼容的高质量数据。此外，我们的方案强调考虑炎症反应的分区，光谱流面板设计和自身荧光光谱分析。这些方法对于提高肺部研究的严谨性至关重要。我们应用该方案对C57BL/6J小鼠肺宿主对甲型流感病毒反应中的白细胞进行精确表征和定位。特别是，我们证明该方案提高了气道内肺泡巨噬细胞的量化和定位。该方法是可修改的和可扩展的，以允许进一步表征白细胞群体的特殊利益。我们描述了一种新的方法组合，包括使用血管内和气管内CD45标记的双重体内区室分析，用于识别淋巴样细胞和非淋巴样细胞的广泛抗体，以及使用细胞自身荧光来帮助解决和识别免疫细胞群的光谱流式细胞术。这种方法可以精确定位可灌洗气道、不可灌洗气道、肺间质组织和肺血管边缘的免疫细胞。
Studies of pulmonary inflammation require unique considerations due to the complex structure and composition of the lungs. The lungs have multiple compartments and diverse immune cell populations, with inherently high autofluorescence, and are involved in the host response to pulmonary pathogens. We describe a protocol that accounts for these factors through a novel combination of methodologies—in vivo compartmental analysis and spectral flow cytometry with a broad panel of antibodies. In vivo compartmental analysis enables the precise localization of immune cells within the marginated vasculature, lung interstitium, nonlavageable airways, and lavageable airways of the lungs, as well as the pulmonary lymph nodes. Spectral flow cytometry with a broad panel of antibodies supports an unbiased exploratory approach to investigating diverse immune cell populations during pulmonary inflammation. Most importantly, spectral flow uses cellular autofluorescence to aid in the resolution and identification of immune cell populations. This methodology enables the acquisition of high-quality data compatible with informed gating and dimensionality reduction algorithms. In addition, our protocol emphasizes considerations for compartmentalization of the inflammatory response, spectral flow panel design, and autofluorescence spectra analysis. These methodologies are critical for increasing the rigor of pulmonary research. We apply this protocol for the precise characterization and localization of leukocytes in the pulmonary host response to influenza A virus in C57BL/6J mice. In particular, we demonstrate that this protocol improves the quantification and localization of alveolar macrophages within the airways. The methodology is modifiable and expandable to allow for further characterization of leukocyte populations of special interest. NEW & NOTEWORTHY We describe a novel combination of methodologies that incorporates dual in vivo compartmental analysis using intravascular and intratracheal CD45 labeling, a broad panel of antibodies for identifying lymphoid and nonlymphoid cells, and spectral flow cytometry that uses cellular autofluorescence to aid in resolving and identifying immune cell populations. This methodology allows precise localization of immune cells in the lavageable airways, nonlavageable airways, interstitial lung tissue, and marginated in the lung vasculature.
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宿主对甲型流感病毒感染的免疫反应
影响因子：
7.3
作者：
Chen X;Liu S;Goraya MU;Maarouf M;Huang S;Chen JL
通讯作者：
Chen JL
DOI：
10.1007/978-1-4939-8570-8_3
发表时间：
2018
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Gibbings SL;Jakubzick CV
通讯作者：
Jakubzick CV
影响因子：
3.2
作者：
Brune, Jourdan E.;Chang, Mary Y.;Frevert, Charles W.
通讯作者：
Frevert, Charles W.
影响因子：
1.7
作者：
Frevert, CW;Warner, AE;Brain, JD
通讯作者：
Brain, JD
影响因子：
2.2
作者：
Barletta, Kathryn E.;Cagnina, R. Elaine;Wallace, Kori L.;Ramos, Susan I.;Mehrad, Borna;Linden, Joel
通讯作者：
Linden, Joel