Electrophysiology on P2X7-HEK cells

HEK293 cells stably transfected with the human P2X7 receptor were grown in DMEM containing 10% FBS, 1% P/S, and 800 μg/ml geneticin (G418). Cells were plated on glass coverslips and grown for 48 h before experimentation. Cells were recorded at room temperature and at a holding potential of –50 mV. Patch pipettes were pulled from borosilicate glass, with resistances of 2–5 MΩ. Patch pipettes contained saline: 130 mM K-gluconate, 10 mM KCl, 0.1 mM CaCl₂, 2 mM Mg-ATP, 1.1 mM EGTA, 10 mM HEPES, and pH 7.4 KOH. Bath saline contained: 140 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgCl₂, 10 mM HEPES, 11 mM glucose, and pH 7.4 NaOH. Data were acquired at 10 kHz with an Axopatch 200B amplifier and analyzed off-line with pClamp 10 software (Molecular Devices). All cell culture materials were purchased from Invitrogen.