Optimized protocols for isolation, fixation, and flow cytometric characterization of leukocytes in ischemic hearts
Optimized protocols for isolation, fixation, and flow cytometric characterization of leukocytes in ischemic hearts
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DOI：
10.1152/ajpheart.00137.2019
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发表时间：
2019-09-01
影响因子：
4.8
通讯作者：
Bansal, Shyam S.
中科院分区：
文献类型：
--
作者：
Covarrubias, Roman;Ismahil, Mohamed Ameen;Bansal, Shyam S.
心肌梗死后的免疫激活是一系列精心安排的细胞反应，以影响组织修复和愈合。然而，过度和失调的炎症可导致左心室重塑和心脏结构和机械属性的病理改变。因此，识别炎症的关键途径和关键细胞介质对于设计心肌梗死和缺血性心力衰竭的免疫调节疗法至关重要。尽管如此，从心脏分离单个核细胞的实验方法是多种多样的，能够在尽可能短的时间内最大限度地获得活细胞的详细方案并不容易获得。在这里，我们描述了心脏CD45(+)白细胞的分离、固定和流式细胞术特征的优化方案。这些方案避免了耗时的冠状动脉灌注和密度介导的细胞分离步骤，导致心脏消化的高细胞产量，没有污染的血管内细胞。此外，与甲醇和丙酮相比，我们发现使用1%多聚甲醛固定细胞是最理想的，因为它不影响抗体结合或细胞形态，从而为研究激活/渗透相关的细胞粒度和大小变化提供了相当大的优势。这些都是高度通用的方法，可以很容易地简化需要从不同组织中同时分离免疫细胞的研究，或者部署在包含大量具有时间敏感性约束的样本的研究中。这些方案被优化以处理多个样本/组织，同时能够在尽可能短的时间内最大限度地获得免疫细胞。结果表明，低速离心法可替代冠状动脉长时间灌流去除血管内细胞，并可通过40目的滤膜代替密度介导法分离单个核细胞，后者通常会导致沉淀物中50-70%的细胞丢失。在流式细胞仪分析中，用1%多聚甲醛固定细胞比用甲醇和丙酮等有机溶剂固定细胞要好。
Immune activation post-myocardial infarction is an orchestrated sequence of cellular responses to effect tissue repair and healing. However, excessive and dysregulated inflammation can result in left ventricular remodeling and pathological alterations in the structural and mechanical attributes of the heart. Identification of key pathways and critical cellular mediators of inflammation is thus essential to design immunomodulatory therapies for myocardial infarction and ischemic heart failure. Despite this, the experimental approaches to isolate mononuclear cells from the heart are diverse, and detailed protocols to enable maximum yield of live cells in the shortest time possible are not readily available. Here, we describe optimized protocols for the isolation, fixation, and flow cytometric characterization of cardiac CD45(+) leukocytes. These protocols circumvent time-consuming coronary perfusion and density-mediated cell-separation steps, resulting in high cellular yields from cardiac digests devoid of contaminating intravascular cells. Moreover, in contrast to methanol and acetone, we show that cell fixation using 1% paraformaldehyde is most optimal as it does not affect antibody binding or cellular morphology, thereby providing a considerable advantage to study activation/infiltration-associated changes in cellular granularity and size. These are highly versatile methods that can easily be streamlined for studies requiring simultaneous isolation of immune cells from different tissues or deployment in studies containing a large cohort of samples with time-sensitive constraints.NEW & NOTEWORTHY In this article, we describe optimized protocols for the isolation, fixation, and flow cytometric analysis of immune cells from the ischemic/nonischemic hearts. These protocols are optimized to process several samples/tissues, simultaneously enabling maximal yield of immune cells in the shortest time possible. We show that the low-speed centrifugation can be used as an effective alternative to lengthy coronary perfusion to remove intravascular cells, and sieving through 40-mu m filter can replace density-mediated mononuclear cell separation which usually results in 50-70% cell loss in the sedimented pellets. We also show that cell fixation using 1% paraformaldehyde is better than the organic solvents such as methanol and acetone for flow cytometric analysis.