Neutrophil Adhesion to Endothelial Cells Modulated by EVs

MLMVECs were pre-stimulated with 100 ng/ml TNF-α for 4 h and rinsed three times with warm HBSS. EVs from different groups were normalized to the same concentration. 1 × 10⁵ neutrophils were incubated with 100 μl EVs (∼10⁸) for 1 h at 37°C with or without the presence of complement inhibitor DAF (10 μg/ml). Neutrophils were then pelleted at 300 g for 5 min, resuspended, and applied on MLMVECs. After 1 h incubation, non-adherent neutrophils were washed off with HBSS for three times (Chatterjee, Yang, Ma, Cha, et al., 2020 (link)). Coverslips were fixed, permeabilized with PBS+0.1% Triton X, and blocked with 10% donkey serum. MLMVECs and neutrophils were labelled with goat anti-mouse ICAM-1 antibody and rat anti-mouse neutrophil antibody respectively overnight at 4°C. After washing three times, donkey-anti-goat AlexaFluor568 and donkey-anti rat AlexaFluor488 were added on the coverslips for 1 h. Prolong Diamond Mounting Medium was used to for nuclei staining and mounting. Images were captured using Leica SP8 Spectral Inverted Laser Scanning Confocal Microscope.