Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes.
Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes.
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DOI：
10.3791/61348
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发表时间：
2020-06-17
期刊：
影响因子：
--
通讯作者：
Lavine JA
中科院分区：
文献类型：
--
作者：
Droho S;Cuda CM;Lavine JA
先天免疫系统在眼部病理生理学中发挥重要作用，包括葡萄膜炎、糖尿病性视网膜病变和年龄相关性黄斑变性。先天免疫细胞，特别是单核吞噬细胞，表达重叠的细胞表面标记，这使得识别这些群体成为一个挑战。多参数流式细胞术可以同时定量分析多种细胞表面标记物，以区分小鼠眼中的单核细胞、巨噬细胞、小胶质细胞和树突状细胞。该方案描述了整个小鼠眼睛的去核、眼部解剖、消化成单细胞悬浮液以及对单细胞悬浮液进行骨髓细胞标记物的染色。此外，我们还解释了使用单色对照确定电压和使用荧光减一对照描绘阳性门的正确方法。多参数流式细胞术的主要限制是缺乏组织结构。这种限制可以通过单个眼室的多参数流式细胞术或互补免疫荧光染色来克服。然而，免疫荧光由于缺乏定量分析以及大多数显微镜上荧光团数量减少而受到限制。我们描述了使用多参数流式细胞术对激光诱导脉络膜新生血管中的单核吞噬细胞进行高度定量分析。此外，多参数流式细胞术可用于转录组或蛋白质组研究中巨噬细胞亚群的鉴定、命运图谱和细胞分选。
The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. Innate immune cells, specifically mononuclear phagocytes, express overlapping cell surface markers, which makes identifying these populations a challenge. Multi-parameter flow cytometry allows for the simultaneous, quantitative analysis of multiple cell surface markers in order to differentiate monocytes, macrophages, microglia, and dendritic cells in mouse eyes. This protocol describes the enucleation of whole mouse eyes, ocular dissection, digestion into a single cell suspension, and staining of the single cell suspension for myeloid cell markers. Additionally, we explain the proper methods for determining voltages using single color controls and for delineating positive gates using fluorescence minus one controls. The major limitation of multi-parameter flow cytometry is the absence of tissue architecture. This limitation can be overcome by multi-parameter flow cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced number of fluorophores on most microscopes. We describe the use of multi-parametric flow cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter flow cytometry can be used for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies.
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