Figure 1.

Microvesicle staining, gating, and quantification. (A) Megamix-Plus side-scatter (SSC) beads (polystyrene) with size references of 0.16, 0.2, 0.24, and 0.5 μm were applied to define a flow cytometric SSC-based gate for microvesicles (MVs) (defined as lipid vesicles). Lipid vesicle equivalents (eq) of the indicated sizes were estimated, taking the different refractive indices of polystyrene and lipid vesicles into account. Gate 1 contains TruCount beads used for quantification of MVs. Gate 2 corresponds to the MV gate used throughout the study. For comparison, normal platelets stained with anti-CD61 antibody were added to the sample (gray events). (B) Forward-scatter (FSC)/SSC characteristics of MVs isolated from culture supernatants and stained with calcein (gray events). (C) MVs contained in culture supernatants from peripheral blood mononuclear cells (PBMCs) incubated with the TLR9-agonist ODN2395. The MVs were incubated with calcein and anti-G3BP antibody (gray) or isotype control (black). (D) Corresponding histogram after staining with anti-dsDNA antibody (gray) or isotype control (black). (E) Contour plot of MVs released from non-stimulated PBMCs and stained for G3BP (x-axis) and dsDNA (y-axis). (F) Corresponding contour plot of MVs released from PBMCs stimulated with ODN2395. Events within gate 2 are shown in (C) through (F).