Af 488 mouse anti human ace2

Antibody solutions were centrifuged at 14,000 × g for 1 h at 4 °C to remove aggregates before use. EVs (1–2 μg EV proteins, as measured on Nanodrop, in 20 μL of PBS) were blocked using 1 μg of mouse serum IgG for 10 min at RT then incubated with: AF-488 mouse anti-human ACE2 (Clone # 171607) (R&D systems, FAB9333G, 0.4 µg/2 µg EVs), APC mouse antihuman CD81 (Clone JS-81 (RUO)) (BD Biosciences, 561958, 1 µL/2 µg EVs), AF-647 mouse antihuman CD63 (Clone H5C6 (RUO)) (BD, Biosciences, 561983, 2 µL/2 µg EVs), AF-488 isotype control mouse IgG2b (Clone # 20102) (R&D systems, IC003G, 0.4 µg/2 µg EVs), APC isotype control mouse IgG₁κ (Clone MOPC-21 (RUO)) (BD Biosciences, 555751, 1 µL/2 µg EVs) or AF-647 isotype control mouse IgG₁κ (Clone MOPC-21 (RUO)) (BD, Biosciences, 557714, 2 µL/2 µg EVs) for 45 min at 4 °C. The solution was then diluted to 200 μL with PBS and the samples were run on Apogee A50 microflow cytometer (MFC) (Apogee Flow Systems, Hertfordshire, UK) (http://www.apogeeflow.com/products.php). The reference ApogeeMix beads (Apogee Flow Systems, 1493), were used to assess the performance of Apogee MFC and to compare the size distribution of the EVs. PBS was run as a background control. Data were analyzed using Flow Jo v10.6.2.

Cells were blocked with mouse serum IgG (Sigma, 15381) for 10 min at room temperature and then incubated with specific antibodies; AF-647 mouse anti-human ACE2 (Clone # 535919) (R&D systems, FAB9332R), AF-488 mouse anti-human ACE2 (Clone # 171607) (R&D systems, FAB9333G) (0.4 µg/10⁶ cells), AF-647 isotype control mouse IgG2b (Clone # 20102) (R&D systems, IC003R) or AF-488 isotype control mouse IgG2bAF488 (Clone # 20102) (R&D systems, IC003G) for 45 min on ice, followed by washing twice with 2% EV-free FBS/PBS. Finally, the cells were diluted in 2% EV-free FBS/PBS and analyzed on a BD-LSR II flow cytometer (BD Biosciences). Data were analyzed by BD FACSDiva softwares v8.0.2 or v8.0.3 or Flow Jo v10.6.2.