CHME-5 cells produce microvesicles (MVs) in response to ATP. (A) Analysis of the surface expression of P2X7-R by flow-cytometry in HEK-293T (left panel) and CHME-5 cells (right panel). Unstained cells (red curves) were used as negative control; stained HEK-293T and CHME-5 cells are represented as blue curves. (B) Electron micrographs showing the plasma membrane of not treated (NT, left panel) and ATP-treated (+ATP, right panel) CHME-5 cells. Multiple round-shaped structures budding from the plasma membrane appeared mainly at the cell surface of ATP-treated cells. The images are representative of two experiments, in which at least 15 fields per condition have been analyzed. The scale bar on the right, valid for both panels, is 2 µm. (C) Scanning electron micrographs of representative CHME-5 cells, untreated (NT, upper panels) or stimulated with 1 mM ATP for 7 min (+ATP, lower panels) are shown. Insets magnifications are shown in right panels. These images are representative of 20 randomly selected fields per condition. (D) Snapshots taken from live-cell recordings of farnesylated-GFP transfected cells exposed to ATP (1 mM, 7 min) are shown; the scale bar at the upper right is 10 µm. The arrows indicate the dynamics of a portion of the plasma membrane during the blebbing/shedding process. The original Video S1 is enclosed into Supplementary Material. (E) A representative cell stained with antibodies against actin (green), surface desmoyokin-AHNAK (dA, red), and flotillin-1 (magenta); 6× magnifications of nascent vesicles are shown in the insets. Scale bar at the upper left is 10 µm. (F) The set up for flow cytometry quantification of MVs is shown: (left panel) FITC-conjugated beads of known dimensions (0.3–0.9 µm) were used as reference for gating. Bona fide MVs would correspond to the IB4+ events comprised into the gate determined by beads positioning inside the plot. Untreated versus ATP-stimulated CHME-5 cells were used as sources of MVs to test the sensitivity of this set up (middle and right panels, respectively), as well as that of tuneable resistive pulse sensing (G) and western blotting for flotillin-1 (H); in the latter a control condition with oxidized ATP (oxATP), a P2X7 inhibitor, was included. Full-length blots are presented in Figure S4 in Supplementary Material.