Preparation of the Murine Anti-Human P2X7 Receptor Monoclonal Antibody (Clone L4)

The murine anti-human P2X7 receptor monoclonal antibody (mAb) (clone L4) has been used to study the expression and function of the P2X7 receptor on primary leukocytes, keratinocytes, osteoblasts and neuronal cells, as well as various cell lines. This antibody has also been used to characterize polymorphic variants and isoforms of the P2RX7 gene and P2X7 site-directed mutations, and to identify molecules coassociated with P2X7 in the plasma membrane. This chapter describes the maintenance and cryopreservation of the L4 hybridoma cell line, as well as the generation of tissue culture supernatant containing the anti-human P2X7 mAb, and its subsequent purification by Protein A chromatography and conjugation to DyLight™ 488. Moreover, this chapter describes flow cytometric assays to assess the blocking activity and binding of the anti-human P2X7 mAb against P2X7 on human RPMI 8226 multiple myeloma cells.