αvβ3 integrin

We cultured GECs and podocytes on six-well plates or chamber slides (Thermo Scientific, Waltham, MA, USA). We incubated cells with PKH26-labelled EVs for 1 h, and then cells seeded on chamber slides were fixed with paraformaldehyde (Sigma Aldrich), nuclei were counterstained in blue by 2.5 μg/mL Hoechst (Sigma Aldrich), evaluated by confocal microscopy (Zeiss LSM 5 PASCAL, Jena, Germany). Cells cultured on six-well plates were detached by EDTA (Sigma) and analyzed by FACS (FACS Calibur, Becton Dickinson, Franklin Lakes, NJ, USA). In selected experiments, PKH26-labelled EVs were pre-incubated with 1 μg/mL of different antibodies directed to block the binding to αVβ-3 integrin (Biolegend, San Diego, CA, USA), α4-integrin, α6-integrin (Chemicon, Temecula, CA), CD29 or L-selectin (Becton Dickinson).

FACS analysis was performed by the Guava easyCyte Flow Cytometer (Millipore, Billerica, MA, United States) and analyzed with InCyte software using the following FITC-, PE- or APC- conjugated antibodies: α4-integrin, α6-integrin (Miltenyi Biotec, Bergisch Gladbach, Germany), β1-integrin, L-selectin (BD Biosciences), αVβ3-integrin (Biolegend, San Diego, CA, USA). FITC, PE or APC mouse isotypic IgG (Miltenyi Biotec) were used as negative controls. Briefly, EPC-derived EVs (5 × 10⁸ particles) were resuspended in 100 µL of 0.1 µm filtered saline solution and incubated with antibodies for 15 min at 4°C; then samples were diluted in 200 µL filtered saline solution and acquired by the instrument.