Figure 3

Cell sorting and high resolution confocal microscopy of EV-immune cell aggregates. To confirm the association of immune cells with EVs, monocytes, granulocytes, and T cells were sorted onto tissue culture slides, fixed, and analyzed by high resolution confocal microscopy as described in the Methods section. Prior to sorting, human whole blood (freshly drawn or stored for 3 h at 37 °C) was stained with lactadherin-AF647 and CD41-AF488 to identify platelet-derived EVs, CD45-PB as leukocyte marker, and either CD14-PE for the sorting of monocytes, CD66b-PE for the sorting of granulocytes, or CD3-PE for the sorting of T cells. (a) Red blood cells and platelets were excluded based on their side scatter (SS) and forward scatter (FS) area characteristics (left panel); SS height vs. SS area dot plots were used to exclude cell aggregates (middle panel), and granulocytes, monocytes, as well as lymphocytes were predefined in dot plots based on SS height vs. CD45 expression (right panel). (b) CD45⁺CD66b⁺ granulocytes, (c) CD45⁺CD14⁺ monocytes, and (d) CD45⁺CD3⁺ T cells were sorted onto 18-well flat bottom µ-slides, fixed, and analyzed by high resolution confocal microscopy; (e) representative images of sorted granulocytes, monocytes, and T cells obtained by high resolution confocal microscopy. Cells and EVs were stained as described above with CD45 (leukocyte marker; blue), CD66b (granulocyte marker; red), CD41 (platelet marker; green), and lactadherin as marker for phosphatidylserine exposing EVs (white). In the overlay, platelet EVs are visualized on the surface of and/or inside granulocytes and monocytes, but not T cells; (f) monocyte associated with platelets, which are clearly distinguished from platelet-derived EVs based on their size and on their exposure of phosphatidylserine. Scale bar, 1 µm.