In order to characterize the molecular pathway/s underlying this newly described cytokine function, we tested the hypothesis that they could enhance the release of MVs by regulating the P2X7 signaling network, at any level. One possibility is that IFN-γ and IL-4 increase the expression of the receptor allowing the cells to be more sensitive to exATP. Time-course experiments show that, starting from 3 h of stimulation both cytokines increased the levels of P2X7, although significantly only at 6 h of IFN-γ treatment (p: 0.0361) (Figure 4A). Additionally, we checked whether this receptor upregulation is also paired to an increase in exATP concentrations, a condition that can be achieved by extensive cell death or by exocytosis of specific ATP-storing granules (27). As IFN-γ and IL-4 seem to be anti-rather than pro-apoptotic factors (at least at the times and concentrations used here), the first possibility was unlikely. Intracellularly, microglial cells accumulate ATP into specialized granules which can be visualized by acridine derivatives such as quinacrine (Figure 4B). In order to check whether cytokines promote ATP exocytosis, cell supernatants were collected and processed for measuring the concentrations of exATP. For preventing exATP degradation, treatments were performed in the presence of an ecto-ATPase inhibitor: no significant differences were observed among the conditions investigated, at any of the time points tested (Figure 4C). The average concentrations of exATP measured here fall within a nanomolar range, which is in line with those previously reported for experiments in which ecto-ATPase inhibitors have been employed (28).