Cells were lysed in Trizol (Invitrogen) and RNA was purified according to the manufacturer’s instructions; residual DNA was removed by DNase treatment at 37°C for 30 min. cDNA synthesis from 5 µg of total RNA was performed using Ready-To-Go You-Prime First-Strand Beads (Amersham) and Random Hexamer (New England Biolabs) according to the manufacturer’s instructions. The levels of P2X7 and GAPDH mRNA was measured by real time RT-PCR (Applied Biosystem); P2X7 (Hs00175721_m1), GAPDH (Hs99999905_m1). The 2−ΔΔCT method was used to calculate relative changes in gene expression as previously described (9).