Characterization of HUVEC-derived apoptotic particles by flow cytometry and fluorescence microscopy. (A) FSC/SSC dot plot analysis of particles from apoptotic HUVECs. Platelets (blue) were used as a size marker (1-4 μm, gate R2). Conditioned medium from apoptotic HUVECs contains dead cells and large cell debris (gate R1), apoptotic bodies (orange, gate R2), and microparticles (green, gate R3). Apoptotic bodies-rich medium (ABRM), obtained after centrifugation (800g, 10 minutes), contains mainly apoptotic bodies and microparticles. Apoptotic bodies-depleted medium (ABDM), obtained after centrifugation (16 000g, 20 minutes) of the ABRM, contains mainly microparticles and some apoptotic bodies. The percentage of events is given in the upper left corner of each region gate. (B) Annexin V/FITC (FL-1) and PI (FL-2) dot plot analysis of live cells, dead cells, and cell-derived particles. Live cells (red) showed an extremely low binding of annexin V and PI. Dead cells, apoptotic bodies, and microparticles (as gated in panel A, black) stain positively with annexin V. Furthermore, dead cells and apoptotic bodies, but not microparticles, stain positive with PI. The quadrant gates were set on the respective unstained control population. The percentage of events is given in the upper right corner of the respective region. (C) Fluorescence microscopy of DAPI⁺, PI⁺, and lectin-FITC⁺ apoptotic bodies (scale bars represent 2.5 μm). Representative plots and images from 3 to 4 independent experiments.