A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues.
A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues.
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DOI：
10.1371/journal.pone.0150606
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Gunn MD
中科院分区：
文献类型：
--
作者：
Yu YR;O'Koren EG;Hotten DF;Kan MJ;Kopin D;Nelson ER;Que L;Gunn MD
流式细胞术广泛用于检查非淋巴组织中的免疫细胞。然而，尚未描述一种既全面又广泛适用的流式细胞术分析方法。我们开发了一种用于非淋巴组织流式细胞术分析的方案，包括组织制备方法、用于细胞染色的 10 荧光染料面板和标准化门控策略，该策略允许同时识别和定量各种正常和发炎非淋巴组织中的所有主要免疫细胞类型。我们证明，我们的基本方案可最大程度地减少细胞损失，可靠地区分巨噬细胞与树突状细胞 (DC)，并识别所有主要的粒细胞和单核吞噬细胞类型。该方案能够准确定量正常肺、心脏、肝脏、肾脏、肠道、皮肤、眼睛和乳腺中的 11 种不同的免疫细胞类型，包括 T 细胞、B 细胞、NK 细胞、中性粒细胞、嗜酸性粒细胞、炎症单核细胞、常驻单核细胞、肺泡巨噬细胞、常驻/间质巨噬细胞、CD11b- DC 和 CD11b+ DC 腺体。我们还表征了几种常用的骨髓和巨噬细胞标记物的表达模式。该基本方案可扩展以识别其他细胞类型，例如肥大细胞、嗜碱性粒细胞和浆细胞样 DC，或对特定细胞类型进行详细的表型分析。在检查原发性和转移性乳腺肿瘤模型时，该方案允许鉴定几种不同的肿瘤相关巨噬细胞表型，其外观对个体肿瘤细胞系具有高度特异性。该协议提供了一个有价值的工具来检查免疫细胞库并跟踪各种组织和实验条件下的免疫反应。
Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b- DC, and CD11b+ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions.