S74 P2X7 anti-P2X5 receptor (#APR-005), and anti-P2X6 receptor antibodies [67]. Once cloned from rat brain, P2X7 was described as the P2Z receptor which is responsible for the ATP-dependent lysis of macrophages through the formation of membrane pores permeable to large molecules and could function also in fast synaptic transmission as a calcium-permeable cation channel [68]. P2X7 channels are reported to be functionally expressed in several types of lymphocytes in which they mediate responses to high extracellular ATP, especially programmed cell death and cytokine secretion. In addition, P2X7 channels are expressed in macrophages and the related brain microglia, where they mediate activation and prolif- eration and therefore are central players in immunogenic responses to inflammation. P2X7 are probably expressed in neurons where they function both as classical ligand-gated ion channels and as mediators of programmed cell death in response to environmental signals. The central role played by P2X7 channels in responses of the immune system have placed it as a promising drug candidate to treat immune system-derived conditions. In such a case, the exact expression pattern and the exact function of the receptor in each tissue are necessary for drug development as a risk assessment. This is the point where specific antibodies, specific pharmacology, and KO techniques are valuable experimental tools. Alomone Labs has generated two anti-P2X7 receptor antibodies, one intracellular (#APR-004 and its ATTO-550 fluorescent-labeled version, #APR-004-AO), targeting the P2X7 C-terminal, which is unique among the family of P2X receptors, and a different antibody, anti-P2X7 receptor (extracellular) targeting an epitope on the extracellular moiety of the channel (#APR-008 and its FITC- conjugated version, #APR-008-F). The design of two different antibodies was of particular importance as the P2X7 channel is encoded by a multi-exon gene and therefore different splice variants of the protein may exist. Both antibodies recognize in Western blot analysis and immunocytochemistry P2X7-transfected cells versus the untransfected counterpart in several different systems such as HEK cells, Xenopus oocytes, etc. [69–74]. The lack of P2X7 expression in KO mice was confirmed using Alomone Labs antibodies in Western blot or immunohistochemistry analyses in the following tissues: salivary gland [73], lung [73], tymocytes [75], peritoneal macrophages [76], bone marrow-derived macrophages [64], microglia [64], astroglia [77], retina [78], ependymal cells along the lateral ventricle of the mouse brain [79], and calvarial osteoblasts (skull bone cells) [80]. Purinergic Signalling (2012) 8 (Suppl 1):S61–S79 In several other tissues, P2X7 immunoreactivity was seen also in KO mice; these include: brain [73, 76] (and see early discussion in [81]), splenic follicles and lymph node [82], and spleen [73]. However, some of these observations were accompanied by the discovery of a novel P2X7 splice variant which was not knocked out by the KO procedure and its mRNA was detected in many tissues including brain [73] or by the notion that lymphocytes from KO mice exhibit enhanced P2X7 responses [82]. P2X7 in the immune system In mouse T lymphocytes, P2X7 expression was assessed by anti-P2X7 receptor (extracellular) antibody in immunohis- tochemical studies on splenic follicles and by Western blot analysis of lymph node [82]. It is worth mentioning that in that particular study, Taylor et al. discovered an enhanced P2X7 activity in lymphocytes from KO mice, which was accompanied by the detection of P2X7 expression in these gene-targeted mice. P2X7 was also detected differentially by Western blot analysis in spleens of wild-type versus KO mice [73], which also demonstrated that a functional P2X7 splice variant with an alternative transmembrane domain 1 escaping gene inactivation in P2X7 KO mice. In mouse tymocytes, P2X7 receptors mediate ATP- dependent non-selective pore formation and cell death. The receptor expression in these cells (from two mouse strains) was confirmed by Western blot analysis with the intracellular antibody and a comparison to parallel cells from P2X7 KO mice in which expression was not detected [75]. The main pathway of P2X7R-mediated thymocyte death was necrosis/lysis rather than apoptosis. Using anti-P2X7 receptor and anti-P2X7 receptor (extra- cellular) antibodies, it was shown by Western blot analysis that the expression of P2X7 receptors in peritoneal macro- phages from control mice is totally absent in similar preparations from KO mice [76]. By using the intracellular antibody in FACS analysis in the same cell type, the expression of the channel was demonstrated. Regarding modulation of the receptor’s activity in relation to cell death, it was shown that nucleoside triphosphate diphos- phohydrolase 1 (NTPDase1)/CD39 (which is the dominant ectonucleotidase expressed by murine peritoneal macro- phages) protects peritoneal macrophages from ATP-induced death via the suppression of P2X7 activation by high extracellular ATP [83]. Co-immunoprecipitation experi- ments in these cells demonstrated that following activation with a “danger signal,” Biglycan, a ubiquitous leucine-rich repeat proteoglycan of the extracellular matrix, P2X7 receptors interact with TLR2/4 in wild-type but not in KO mice to activate interleukin 1β secretion [84]. In experiments that ruled out the expression of P2X7 in human neutrophils, the mouse macrophage cell line J774