Visualizing Leukocyte-Endothelial Interactions

Leukocyte-endothelial interactions along the carotid artery were analyzed in Apoe^−/−Cx₃cr1^GFP reporter mice by intravital microscopy as previously reported^{3 (link)–5 (link)}. In brief, mice were placed in supine position, and the right jugular vein was cannulated with a catheter for antibody injection. Intravital microscopy was performed after injection of a PE-conjugated antibody to Ly6G (1 μg; clone 1A8; BioLegend) and 4’,6-Diamidino-2-phenylindol (DAPI, Thermo Fischer). Using an Olympus BX51 microscope equipped with a Hamamatsu 9100–02 EMCCD camera, and a 10× saline-immersion objective movies of 30 s were acquired and analyzed offline. In the carotid artery, one field of view was analyzed per mouse. gfp expressing cells were considered monocytes. For identification of NET like structures, the original DAPI image was transformed into a 8 bit gray scale image and subsequently thresholded. Particles in the latter image were quantified and particles above 80 px^{2 (link)} and a circularity below 0.75 were considered NET like structures.