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HEK cells transiently transfected with P2X receptor were seeded overnight at 40,000 cells/100uL/well in a Costar black wall/clear bottom 96-well plate. The cells were incubated with 100 uL of the MP dye-loading solution in a 5% CO2, 37°C incubator for 60 minutes. ATP (50 uL/well) was added by FlexStation to achieve the final indicated concentrations. The fluorescence signal was measured with bottom read mode at Ex/Em = 620/650 nm (cutoff at 630 nm).