Annexin 5 fluorescein propidium iodide fitc pi co staining

Human peripheral blood mononuclear cells (PBMC) were isolated from four healthy male volunteers by venous blood draw and density gradient centrifugation with Ficoll-Paque (GE Healthcare Bio-Sciences AB, Sweden). PBMCs (25 × 10⁶ cells/ml) were resuspended in serum-free medium (CellGro, CellGenix, Freiburg, Germany). An automated cell counter (Sysmex Inc., USA) was used to determine cell count. PBMCs were gamma-irradiated with 60 Gy to induce apoptosis. Induction of apoptosis was confirmed by annexin V-fluorescein/propidium iodide (FITC/PI) co-staining (Becton Dickinson, Franklin Lakes, NJ, USA) using a flow cytometer. At 20h after irradiation 58% of PBMCs were annexin V/PI positive (supplementary Fig. S1). CM was collected from cultures at 2, 4, and 20 h time points, and then, centrifuged (500 × g for 9 min) to remove cell debris. CM was stored at −80 °C for subsequent protein and lipid analyses. Fresh CM was used for microparticle and exosome separations.

The induction of apoptosis was measured by annexin V-fluorescein/propidium iodide (FITC/PI) co-staining (Becton Dickinson, Franklin Lakes, NJ, USA) using a flow cytometer as described previously [32 (link)].