Fig. 6

EVs from endothelial cells bind to leukocytes in vitro. a–c EVs collected from supernatants of TNF-α-treated BMEC cultures were incubated with isolated PBLs. a z-stack confocal images show PKH-67-labeled (green) exosome- and microvesicle-size EVs separated by differential ultracentrifugation can bind to PKH-26-labeled naïve PBLs (red), along with DRAQ5 staining of PBL nuclei (blue). Insets highlight co-localization of staining on a single PBL. b–c Binding of total PKH-67-labeled EVs to naïve PBLs. FACS analysis revealed 4.5% of the PBLs were PKH-26⁺ PKH-67⁺ (double-positive), suggestive of binding. The percentage of PBLs binding to PKH-67⁺ EVs diminished by 93%, while the PKH-67 signal intensity per leukocyte (mean fluorescence intensity), a reflection of the relative EV binding/cell, decreased by 65% in the presence of a 500-fold excess of unlabeled EVs. d Total eGFP-CLN-5⁺ EVs, FACS-purified from blood plasma of Tie-2-eGFP-CLN-5 mice at D8 EAE, bind to naïve PBLs in vitro. In this case, PBLs are identified by CD45 staining (blue). Inset further highlights a punctate binding pattern of eGFP-CLN-5⁺ EVs to PBLs