Cd63 antibody

The anti‐hACE2 Alexa Fluor 488 antibody used for flow cytometry evaluation of ACE2 expression was obtained from R&D Systems (Catalog no. FAB 9332G). The rabbit‐anti‐SARS‐CoV‐2 spike neutralizing antibody was obtained from Sino Biological (Catalog no. 40592‐R001). The normal rabbit IgG isotype control Ab was obtained from R&D Systems (Catalog no. AB‐105‐C). The calnexin antibody for western blot was obtained from Abcam (Catalog no. AB22595). The polyclonal anti‐spike (S2) antibody was obtained from Abcam (Catalog no. AB272504). The CD63 antibody was obtained from Abcam (Catalog no. AB59479).

Dose dependent binding of exosomes to type I collagen was analyzed using ELISA. 96-well assay plates were coated with 5 μg of type I collagen per well. The coated plates were incubated for 1 hour at room temperature with increasing volumes of exosomes. The plates were then washed 3 times in PBS, fixed using 4% neutral buffered formalin, permeabilized using PBS containing 0.5% triton x-100, and blocked for 1 hour at room temperature with PBS containing 5% BSA. The wells were then incubated for 1 hour at room temperature with CD63 antibody (Abcam, 1/1000 dilution), washed 3 times with PBS, and incubated for 1 hour with HRP conjugated secondary antibody (1/3000 dilution). All antibody dilutions were performed in PBS containing 5% BSA. Turbo TMB ELISA substrate was used to for the colorimetric assay followed by addition of acid stop solution (1 M sulfuric acid). The absorbance at 495 nm was measured using a Bio-tek ELISA plate reader. The experiment was performed in quadruplicate. The absorbance was normalized to the control wells (no exosome added, but containing type I collagen and treated with both primary and secondary antibodies) and the results were plotted graphically with volume of exosome on the x-axis and normalized absorbance units on the y-axis.