Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a ACE2+ EVs detected in human plasma samples of sero-negative controls (light blue), acute phase (dark green), and convalescent COVID-19 patients (green). One-tail t test (* p = 0.038, ** p = 0.0061 and ** p = 0.0016). Data are presented as mean values ± SEM. b Representative microflow vesiclometry (MFV) plots with gated ACE2+ EVs from sero-negative, acute phase and convalescent COVID-19 patients. c MFV detection of circulating ACE2 + EVs with CD63 + EVs in human plasma of convalescent COVID-19 patient samples (CSB-029 and CSB-023) (green line). Blue line is isotype IgG-negative control. d Flow profiles of ACE2 expression in HEK and HeLa parental control cells (Con, light blue line, ACE2 − ) and with ACE2 overexpression (ACE2, green line). e NanoSight NTA analysis of the sizes of HEK-derived ACE2 − (ev1Con) and ACE2 + (ev1ACE2) and HeLa-derived ACE2 − (ev2Con) and ACE2 + (ev2ACE2). f Immunoblots of HEK and HeLa (ACE2 − and ACE2 + ) EVs and cell lysates for ACE2, TSG101, CD63, CD81, GRP94 and loading control of the membrane proteins upon Ponceau staining. RIPA buffer and Bradford protein assay were used for cells/EVs lysis and protein measurement, respectively ( N = 1 experiment). g Cryo-EM images of HEK-derived EVs, ACE2 − (evCon, left) and ACE2 + (evACE2, right), stained with ACE2 (top) and CD81 (bottom). Scale bars = 100 nm. h Quantified counts of Apogee MFV-based total extracellular vesicles (EVs) and ACE2 + EVs ( N = 2 experiments with n = 6 technical replicates for total EV particles and n = 3 technical replicates for ACE2 + counts). Control EVs are in light blue and ACE2 + EVs in green. Data are presented as mean values +/− SD. i Overlay flow profiles of ACE2 positivity within CD63 + (left column) and CD81+ (right column) EVs isolated from HEK-ACE2 (top row) and HeLa-ACE2 (bottom row) cells, respectively ( n = 3 technical replicates). Light blue line for Control EVs and green line for ACE2 + EVs.

Article Snippet: 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/10 6 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.

Techniques: Clinical Proteomics, Negative Control, Expressing, Control, Over Expression, Derivative Assay, Western Blot, Membrane, Staining, Bradford Protein Assay, Lysis, Cryo-EM Sample Prep, Isolation