Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach.
Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach.
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DOI：
10.3791/61590
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发表时间：
2020-09-22
期刊：
影响因子：
--
通讯作者：
Sengupta S
中科院分区：
文献类型：
--
作者：
Issah Y;Naik A;Tang SY;Forrest K;Theken KN;Sengupta S
昼夜节律是指各种生物过程以24小时为周期发生的振荡。在分子水平上，这种节律由核心时钟基因的转录 - 翻译反馈回路（TTFL）网络组成。各个组织和器官系统，包括免疫系统，都有自己的生物钟。在体循环中，CD45⁺细胞群的各种成员在一天中会发生波动；然而，在组织驻留的CD45⁺白细胞群中，许多这种节律并不相同，甚至不相似。在研究肺部炎症的昼夜节律调节作用时，可能需要对肺内的CD45⁺进行研究。然而，尽管有优化的灌注方法，从循环中截留的白细胞仍会在肺中存在。设计该实验方案的目的是区分血管内和实质内的白细胞。为此，在获取肺组织前不久，通过颈静脉向小鼠注射一种荧光标记的CD45抗体。之后，使用一种定制的肺消化技术对肺进行消化，以获得单细胞悬液。对样本进行实质内免疫细胞常规抗体组合（包括另一种CD45抗体）染色。流式细胞术分析清晰地阐明了细胞群。因此，在肺内和循环免疫细胞在数量和功能上存在差异的情况下，标记和定义肺内CD45⁺细胞的方法将尤为重要。
Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of a web of transcriptional-translational feedback loops (TTFL) of core clock genes. Individual tissues and organ systems, including the immune system, have their own clock. In the systemic circulation, various members of the CD45+ population oscillate across the day; however, many of these rhythms are not identical or even similar in the tissue resident CD45+ leukocyte population. When studying the role of circadian regulation of lung inflammation, CD45+ within the lung may need to be investigated. However, despite optimized perfusion methods, leukocytes trapped from the circulation persist in the lungs. The goal in designing this protocol was to distinguish between intravascular and intraparenchymal leukocytes. Towards this end, mice are injected with a fluorescent tagged CD45 antibody intrajugularly shortly before lung harvest. Thereafter, the lung is digested using a customized lung digestion technique to obtain a single cell suspension. The sample is stained for the regular panel of antibodies for intraparenchymal immune cells (including another CD45 antibody). Flowcytometric analyses shows a clear elucidation of the populations. Thus, the method of labeling and defining intrapulmonary CD45+ cells will be particularly important where the behavior of intrapulmonary and circulating immune cells are numerically and functionally distinct.
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影响因子：
82.9
作者：
通讯作者：
--
全天候免疫。
DOI：
10.1126/science.aah4966
发表时间：
2016-11-25
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Man K;Loudon A;Chawla A
通讯作者：
Chawla A
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：
16.6
作者：
Sengupta, Shaon;Tang, Soon Y.;FitzGerald, Garret A.
通讯作者：
FitzGerald, Garret A.
DOI：
10.1073/pnas.1408886111
发表时间：
2014-11-11
影响因子：
11.1
作者：
Zhang, Ray;Lahens, Nicholas F.;Hogenesch, John B.
通讯作者：
Hogenesch, John B.