Figure 2

Transfer of cytosolic components from the cellular cytosol into E‐GPMVs. A) Left column: Top) Schematic representation of HepG2 cells with enhanced membrane and cytosolic content. Central) Illustration of the formation of equipped‐GPMV (E‐GPMV) from the donor cells. Bottom) Isolated E‐GPMV. Middle column: Top) CLSM micrograph of HepG2 cells containing Chol‐PEG5000‐FITC copolymer in the membrane (Green), Scale bar = 15 µm. Central) CLSM micrograph of E‐GPMVs formation by several cells, Scale bar = 40 µm; Chol‐PEG5000‐FITC copolymer is inserted both in the cells and in the GPMVs membrane. Bottom) CLSM micrograph with a zoom of one isolated E‐GPMV containing Chol‐PEG5000‐FITC copolymer in the membrane (Scale bar = 10 µm). Right column: Top) CLSM micrograph of HepG2 cells simultaneously containing Actin‐RFP protein (Red) and the membrane protein Lck‐GFP (Green), Scale bar = 15 µm. Central) CLSM micrograph of E‐GPMVs formation by several cells, Scale bar = 40 µm; Actin‐RFP protein (Red) and the membrane protein Lck‐GFP (Green) are located both in the cells and in the GPMVs. Bottom) CLSM micrograph with a zoom of one isolated E‐GPMV containing Actin‐RFP protein (Red) and the membrane protein Lck‐GFP (Green), Scale bar 10 µm. CLSM Controls are available in the Supporting Information. B) Flow Cytometry analysis of E‐GPMVs, size characterization by sideward and forward scattering analysis. The ellipse represents E‐GPMV populations (2.0–9.9 µm) used for analysis by flow cytometry. Blue to red color‐coded polystyrene particles in sizes from 2.0, 3.4, 5.11, 7.6, 9.9, and 14.3 µm. Green: E‐GPMV population. C) Size distribution of the E‐GPMVs with membranes supplemented with the membrane protein Lck‐GFP obtained by analysis of CLSM micrographs. N = 501 individual E‐GPMVs. D) Flow cytometry analysis of E‐GPMVs equipped with Chol‐PEG5000‐FITC copolymer (Green) and control (unmodified) GPMVs (Gray). E) Flow cytometry analysis of Annexin V‐Atto488 binding to E‐GPMVs equipped with the copolymer Chol‐PEG5000: Control GPMVs without Annexin V (Red), control GPMVs with added Annexin V (Green) and E‐GPMVs equipped with Chol‐PEG5000 copolymer and added Annexin V (Orange). F) Size distribution of E‐GPMVs equipped with Chol‐PEG5000‐FITC copolymer obtained by analysis of CLSM micrographs. N = 501 individual E‐GPMVs.