Ion channels play essential roles in immune cells, inflammation and immunity (Feske et al., 2015). Among these is the P2X4 receptor, which belongs to the P2X family of purinergic receptors. P2X receptors are trimeric ligand-gated ion channels activated by adenosine 5′-triphosphate (ATP) and other nucleotides (Illes et al., 2021). Activation of P2X receptors causes the rapid flux of Ca2+, Na+ and K+ across the plasma membrane, with P2X4 having the greatest permeability to Ca2+ among all P2X receptors (Stokes et al., 2017). Repeated or prolonged P2X4 activation can also lead to the uptake of organic cations, including fluorescent dyes such as YO-PRO-12+ (Stokes et al., 2017), although this phenomenon is not widely reported. P2X4 is located on the cell-surface membrane and in intracellular compartments, being most abundant in lysosomes, with cell activation altering the distribution of this receptor depending on the cell type or activation stimulus (Kanellopoulos et al., 2021). P2X4 is most reported as a homomeric receptor, but heteromeric receptors containing P2X4 subunits with P2X7 or other P2X subunits have been described (Kanellopoulos et al., 2021; Stokes et al., 2017) and are not discussed further in this article.

P2X4 is present in various cell types including immune cells and has well-established roles in the nervous system (Sophocleous et al., 2022), including pain (Sophocleous and Sluyter, 2023), and in the cardiovascular system (Bragança and Correia-de-Sá, 2020). P2X4 also functions in inflammation and immunity, with emerging roles in sepsis (Antonioli et al., 2019), allergy (Matsuoka et al., 2022), inflammatory bowel disorders (Sluyter, 2023), and other inflammatory and immune disorders including those relating to the central nervous system such as multiple sclerosis (Domercq and Matute, 2019) and amyotrophic lateral sclerosis (Volonté et al., 2023).

This review aims to provide an overview of the techniques commonly used to study P2X4 in immune cells, focusing on those methods used to assess P2RX4 gene expression, the P2X4 protein, and P2X4 ion channel activity in these cells. It is beyond the scope of this article to discuss the many techniques used to study downstream effects of P2X4 activation in isolated immune cells (or those in animal models), which are various and many, and most of which are commonly used by immunologists and others to study immune cell functions such as phagocytosis, cytokine and prostaglandin release, and cell proliferation and migration.