YO-PRO-1 uptake assay

Human embryonic kidney (HEK293) cells were maintained in DMEM medium (Thermo Fisher Scientific) supplemented with 10% FBS (Atlanta Biologicals, Flowery Branch, GA), and 10 μg/mL gentamicin (Thermo Fisher Scientific). HEK293 cells were split into six well plates at a cell density of 2.0 × 10⁶ cells/well and incubated overnight. Three hours after transfection with 2 μg of the full length pdP2X7 in pIM2 vector (IRES-mCherry; modified from pIE2 vector) using jetPRIME reagent (Polyplus-transfection, France), cells were trypsinized, transferred into poly-D-Lysine coated black-walled 96 well plates (Corning, Corning, NY) at 7.5 × 10⁴ cells/well, and incubated for 24 hr. Cells were washed with assay buffer (2 mM KCl, 0.1 mM CaCl₂, 13 mM Glucose, 147 mM NaCl, 10 mM HEPES (pH 7.3)) and were incubated with 5 μM YO-PRO-1 Iodide (Thermo Fisher Scientific), in the presence or absence of antagonists at multiple concentrations at 37°C for 10 min. Upon application of 1 mM ATP (final), uptake of YO-PRO-1 was recorded with 1 min intervals by following the fluorescence change using a Synergy HT multi-detection microplate reader (Bio-Tek, Winooski, VT; Ex: 485 nm/20, Em:528 nm/20, sensitivity 60). To obtain the dose dependent uptake inhibition by antagonists, the initial rates of YO-PRO-1 uptake were plotted against multiple concentrations of the P2X7 specific drugs. The inhibition curves from five independent measurements were fitted to the Hill equation using Origin software (Originlab, Northampton, MA) to determine the IC₅₀ values.