a–h, EVs isolated from the human plasma by the SEC method were incubated with PBS, FITC-LPS or biotin-LPS as indicated at 37 oC for 45 min, and the LPS binding was assessed by the LAL (a,e) and HEK-Blue TLR4 reporter cell (b,f) assays and ImageStream flow cytometry (c,d,g,h) (n=3). i–p, EVs isolated from bEnd.3 endothelial (i–l) and HeLa (m–p) cells cultured in serum-free conditions by the UC method were incubated with PBS or FITC-LPS (500 μg) as indicated at 37 oC for 45 min followed by re-purification via SEC, and the LPS binding was assessed by the LAL (i,m) and HEK-Blue TLR4 reporter cell (j,n) assays and ImageStream flow cytometry (k,l,o,p) (n=3). Combined data from three experiments (a–p) are shown. Each circle represents an independent experiment, and the horizontal lines represent the mean. P values were determined by unpaired two-tailed t-test (a–p).