The Immune Synapse - Methods and Protocols
The Immune Synapse - Methods and Protocols
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免疫突触 - 方法和方案
DOI：
10.1007/978-1-0716-3135-5_26
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Ambrose A
中科院分区：
文献类型：
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作者：
Ambrose A
在这里，我们描述了一种方法，我们称之为“阴影成像”，来分析免疫突触或其他细胞接触处的单个细胞的分泌。在富含配体的载玻片上形成免疫突触和激活细胞后，使用脉冲免疫荧光染色针对富含配基的载玻片表面的蛋白质记录每个细胞的位置。脉冲染色不会穿透突触裂隙，导致在细胞分离后保留的未标记区域或细胞下的“阴影”。分泌的成分，如穿孔素、外切体或其他类型的细胞外小泡，被保留在载玻片上，并可以使用免疫荧光在单细胞基础上进行分析。识别单个细胞分泌不同组合的颗粒、蛋白质和囊泡的能力使我们能够更好地了解免疫细胞分泌的异质性，并可作为一种新的细胞群体表型鉴定方法。
Here, we describe a method, which we term “shadow imaging,” to analyze the secretions of individual cells at immune synapses or other cell contacts. Following immune synapse formation and cellular activation on ligand-rich slides, the position of each cell is recorded using a pulsed immunofluorescence stain against the proteins on the ligand-rich slide surface. The pulsed stain does not penetrate the synaptic cleft, resulting in an unlabeled region or “shadow” beneath cells that is retained following cellular detachment. The secreted components, such as perforin, exosomes, or other types of extracellular vesicles, are retained on the slide and can be analyzed on a single-cell basis using immunofluorescence. The ability to identify single cells secreting different combinations of particles, proteins, and vesicles enables us to better understand the heterogeneity in immune cell secretions and can be used as a novel approach for phenotyping cell populations.