Medium 200 lsgs

Human umbilical vein endothelial cells (HUVECs) were purchased from Cell Applications, cultured in Medium 200 + LSGS (Gibco, Waltham, MA, USA) on a gelatin-coated surface, and Passage 4 cells were used in experiments. Endothelial cells were exposed to RPMI serum-free medium (Gibco) or Medium 200 + LSGS (depleted of vesicles) for 4 h to produce apoptotic-cell- or healthy-cell-conditioned medium respectively as described previously [13 (link), 15 (link), 18 (link), 19 (link)]. To generate fluorescence-emitting vesicles, HUVECs were stained using CellTracker Orange-CMRA dye or SYTO RNASelect Green Fluorescent cell stain (Molecular Probes, Waltham, MA, USA) for 15 min before treatment. Cytochalasin D, monodansylcadaverine, methyl-β-cyclodextrin (MβCD), and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were purchased from Sigma (Burlington, MA, USA). FITC-Transferrin was obtained from Thermo Fisher (Waltham, MA, USA), and annexin V was obtained from BioLegend (San Diego, CA, USA).

Human umbilical vein endothelial cells (HUVEC) were purchased from Cell Applications, cultured in EGM-2MV complete medium (Lonza) or Medium 200 + LSGS (Gibco, Waltham, MA, USA) on a gelatin-coated surface and used at passage 4. To produce a conditioned medium containing ApoExo, cells were exposed to RPMI serum-free medium (Gibco) for 4 h to induce apoptosis in endothelial cells. Serum starvation is a classical inducer of apoptosis. We showed previously that serum starvation in endothelial cells increases chromatin condensation in the absence of cell membrane permeabilization along with caspase-3 activation^{11 (link)–13 (link)}.