Lymphocyte encounters

with interendothelial junctions were determined by following the track of each lymphocyte on the videomicrographs over the characteristic phase-bright band between adjacent EC. In a second technique, lymphocytes were stained with CellTracker Orange according to the manufacturers instructions, then were made to interact with HUVEC monlayer in the parallel-plate flow chamber. After 10 min of shear stress application, the chamber was disassembled, and the cells were stained for VE-cadherin. To study diapedesis, the location of each lymphocyte relative to VE-cadherin staining was analyzed using a LSM 510 confocal microscope (Zeiss, Toronto, Ont., Canada) set to acquire images at 0.4 μm intervals in the z-plane. Lymphocytes were considered

to be associated CT99021 with gap formation in the AJ if a break in endothelial VE-cadherin staining at least 2 μm wide was directly superimposed on the lymphocyte footprint. Lymphocytes were scored by blinded observer for the relationship in the z-plane to the VE-cadherin signal. To study the PECAM-1 enrichment around lymphocytes in the process of diapedesis, PECAM-1bright naïve T cells (CD45RA+) cells were depleted using CD45RA TAC (StemCell Technologies). The cells were stained with CellTracker Blue and were made to interact with the HUVEC monlayer in the parallel-plate flow chamber. After 10 min of shear stress application, the chamber was disassembled, and the cells were double stained for VE-cadherin and PECAM-1. Confluent HUVEC Celastrol monolayers seeded on Matrigel-coated selleck chemical glass coverlips were treated with either DMSO or ND. Cells were fixed,

permeabilized, and blocked as described previously 46. The cells were then double-stained using anti-β-tubulin and anti-VE-cadherin primary and fluorophore-conjugated secondary antibodies. To determine MT and AJ morphology in cells treated with non-silencing or IQGAP1 RNAi, transfected HUVEC were trypsinized and seeded on coverslips at confluency. The monolayer was stained with either β-catenin or double-stained for MT and VE-cadherin. MT density adjacent to AJ was measured using image analysis software (OpenLab, Lexington, MA, USA). Regions of interest were defined extending 3 μm into the cell cortex from VE-cadherin-positive junctions to quantitate MT staining intensity in at least 30 cells in each experiment. To evaluate F-actin cytoskeleton changes, confluent HUVEC monolayers were fixed and permeabilized and F-actin was stained by FITC-phalloidin. To determine the effect of TNF-α treatment and shear stress on junction staining, HUVEC were treated with TNF-α and subjected to shear stress in conditions as described for TEM assay but with no lymphocytes. Then cells were fixed and permeabilized and stained for VE-cadherin, PECAM-1, and Jam-1. CD99 was stained without permeabilization.