Abstract：
The P2X4 receptor is a trimeric ligand-gated ion channel activated by adenosine 5\'-triphosphate (ATP). P2X4 is present in immune cells with emerging roles in inflammation and immunity, and related disorders. This review aims to provide an overview of the methods commonly used to study P2X4 in immune cells, focusing on those methods used to assess P2RX4 gene expression, the presence of the P2X4 protein, and P2X4 ion channel activity in these cells from humans, dogs, mice and rats. P2RX4 gene expression in immune cells is commonly assessed using semi-quantitative and quantitative reverse-transcriptase-PCR. The presence of P2X4 protein in immune cells is mainly assessed using anti-P2X4 polyclonal antibodies with immunoblotting or immunochemistry, but the use of these antibodies, as well as monoclonal antibodies and nanobodies to detect P2X4 with flow cytometry is increasing. Notably, use of an anti-P2X4 monoclonal antibody and flow cytometry has revealed that P2X4 is present on immune cells with a rank order of expression in eosinophils, then neutrophils and monocytes, then basophils and B cells, and finally T cells. P2X4 ion channel activity has been assessed mainly by Ca2+ flux assays using the cell permeable Ca2+-sensitive dyes Fura-2 and Fluo-4 with fluorescence microscopy, spectrophotometry, or flow cytometry. However, other methods including electrophysiology, and fluorescence assays measuring Na+ flux (using sodium green tetra-acetate) and dye uptake (using YO-PRO-12+) have been applied. Collectively, these methods have demonstrated the presence of functional P2X4 in monocytes and macrophages, microglia, eosinophils, mast cells and CD4+ T cells, with other evidence suggestive of functional P2X4 in dendritic cells, neutrophils, B cells and CD8+ T cells.
摘要：
P2X4受体是由5'-三磷酸腺苷(ATP)激活的三聚体配体门控离子通道。P2X4存在于免疫细胞中，在炎症和免疫中具有新兴作用，和相关疾病。这篇综述旨在概述免疫细胞中P2X4的常用研究方法。重点介绍用于评估P2RX4基因表达的方法，P2X4蛋白的存在，这些人类细胞中的P2X4离子通道活性，狗,老鼠和老鼠。免疫细胞中的P2RX4基因表达通常使用半定量和定量逆转录酶-PCR评估。免疫细胞中P2X4蛋白的存在主要使用免疫印迹或免疫化学的抗P2X4多克隆抗体进行评估。但是这些抗体的使用，以及用流式细胞术检测P2X4的单克隆抗体和纳米抗体正在增加。值得注意的是,使用抗P2X4单克隆抗体和流式细胞术显示，P2X4存在于免疫细胞中，在嗜酸性粒细胞中的表达排序，然后是中性粒细胞和单核细胞,然后是嗜碱性粒细胞和B细胞,最后是T细胞。P2X4离子通道活性主要通过Ca2通量测定进行评估，使用细胞可渗透的Ca2敏感染料Fura-2和Fluo-4和荧光显微镜，分光光度法，或流式细胞术。然而,其他方法，包括电生理学，并应用了测量Na通量（使用绿色四乙酸钠）和染料吸收（使用YO-PRO-12）的荧光测定法。总的来说，这些方法已经证明在单核细胞和巨噬细胞中存在功能性P2X4，小胶质细胞,嗜酸性粒细胞,肥大细胞和CD4+T细胞，其他证据表明树突状细胞中P2X4的功能，中性粒细胞，B细胞和CD8+T细胞。
