The antagonist activity of the 17 compounds (applied at 10 μM) was assayed initially on 1321N1 astrocytoma cells stably expressing human P2X4 receptors by measuring the ATP-induced (applied at 1.2 μM) increase in Fluo-4 fluorescence due to calcium influx via P2X4, using 2 μM BX430 as a positive control (Figure 1A). Here, P2RX4 is linked to astrocytoma (excluding glioblastoma).