Pe conjugated anti human cd3

The effects of defibrotide on leucocyte adhesion on endothelial cells were analysed using a parallel‐plate perfusion chamber. SK‐Hep1 monolayers were treated previously with 100 µg/mL defibrotide for 24 hours or left untreated and then grown for 48 hours in medium supplemented with 20% pooled sera of aGvHD patients (n = 15, Table 1) or of healthy donors (n = 15). Then, SK‐Hep1 cells were perfused with control citrated blood from a healthy donor at a shear rate of 300 s⁻¹ for 10 minutes. After perfusion, coverslips were washed with PBS and fixed in 3% paraformaldehyde in PBS. For leucocyte immunostaining, coverslips were treated with 1% BSA, permeabilized with 0.1% Triton‐X and stained with an anti‐human CD45 antibody (Abcam), followed by Alexa488‐conjugated anti‐rabbit secondary antibody (Life Technologies, Carlsbad, CA), and PE‐conjugated anti‐human CD3 (BD Biosciences, San Jose, CA) and DAPI (Sigma‐Aldrich) for nuclei counterstaining. Finally, coverslips were visualized by fluorescent microscopy (Leica‐DM4000B) through a video camera (Leica‐DFC310FX) and analysed using ImageJ software (National Institutes of Health, Bethesda, MD). The number of adherent leucocytes and T cells was expressed as percentage related to the total number of endothelial cells (number of leucocytes per 100 endothelial cells).