Methods for studying P2X4 receptor ion channels in immune cells

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.