a,b, Immunoblotting (a) and negative staining TEM (b) of unshaved EVs and EVs subjected to surface protein shaving with trypsin (shaved EVs). c,d, Unshaved and shaved EVs incubated with FITC-LPS at 37 oC for 45 min were subjected to NTA (c), and the HEK-Blue TLR4 reporter cell assay (d). e,f, Percentage of FITC-LPS+ve EVs (e) and FITC MFI (f) of EVs isolated from FITC-LPS-injected mice and subjected or not to surface-protein shaving (n=3). Combined data from three (c,d) or four (e,f) experiments or one experiment representative of two (a,b) are shown, and horizontal lines represent mean (c–f). P values were determined by unpaired two-tailed t-test.