Fig. 2
An example of a light scatter plot (a) and fluorescence dot plots (b–g) derived from the microvesicle (0.2–1 μm size) gate of the digital flow cytometer analysis of cell-free culture media from TNFα (20 ng/ml) stimulated HBMEC for 20 h. The size gates were determined based on calibration size (0.2–2 μm) beads and with truCOUNT™ (4.2 μm size) beads (a). The positive and negative counts of microvesicles were separated by fluorescence dot plots derived from microvesicle gate of scatter plot of phycoerythrin- and fluorescein (FITC)-conjugated isotype control antibodies (b) and selected candidate cell membrane marker antibodies (c, PECAM-1; d, Integrin α˅β3; e, ICAM-1; f, E-selectin) plus recombinant annexin-V for surface phosphatidylserine and MCAM plus VE-cadherin (f) antibodies stained microvesicles. The example separation of single (+/− (Q1) or −/+ (Q4)), double (+/+ (Q2)) marker positive, and both marker negative (−/− (Q3)) microvesicles is shown in c