fig3

Figure 3. Transient expression of Nef.GFP in h-microglia stimulates extracellular release of smaller vesicles carrying Nef.GFP. Using simple ultracentrifugation, crude total EVs were enriched from media of non-transfected h-microglia cultures (control D2) or 48 h post transfection (Nef.GFP). Representative transmission electron microscopy images of negatively stained pelleted particles from the (A) Nef.GFP and (B) control D2 samples. Scale bars: 1 µm (left) and 200 nm (right). Arrows indicate single extracellular particles in higher magnification; (C) Representative SIM image of pelleted particles in the Nef.GFP sample, attached to the coverslip surface and observed in super-resolution mode; scale bar, 5 µm. One of the Nef.GFP-positive EVs (green; framed in center) is magnified in the top right corner; inset scale bar: 0.5 µm; (D) Frequency (%) plot of diameter distributions (2r, nm) for 1619 morphologically distinct Nef.GFP‑positive EVs. The mean EV 2r (x₀ ± s.e.) is displayed above the plot; (E) Normalized AF4-MALS fractograms recorded by 90° LS detector (solid lines) for pelleted particles from the control D2 (gray) and Nef.GFP (green) samples with displayed root-mean-square radius, R_rms (filled circles). (F) Normalized AF4-MALS fractograms recorded by 90° LS detector (solid lines) for pelleted particles from the control D2 (gray) and Nef.GFP (green) samples, with particle number density per mL (filled circles) as a function of elution time. AF4-MALS: Asymmetric-flow field-flow fractionation coupled to a multi-angle light-scattering detector; SIM: structured illumination microscopy; Nef.GFP: Nef green fluorescent protein; h-microglia: human microglia; EV: extracellular vesicle; LS: light scattering; R_rms: root-mean-square radius; D2: day 2 post-transfection.