Figure 3.

Adherent and crawling neutrophils on apical surface of TNFα-treated endothelium. (A) Crawling time (in seconds) of neutrophils from adhesion to diapedesis was determined for neutrophils that crossed either shCTRL or shADAM10-treated EC monolayers. (B) Velocity (µm/s) and (C) distance (µm) are determined on neutrophils that were on top (apical) of the endothelium that was silenced for ADAM10 (shADAM10) or treated with shCTRL. (D) Velocity (µm/s) and (E) distance (µm) are determined on neutrophils that were underneath (basolateral) of the endothelium that was silenced for ADAM10 (shADAM10) or treated with shCTRL. (F) Time (in seconds) of actual diapedesis measured for neutrophils that crossed either shCTRL or shADMA10-treated endothelial cell monolayers. Data are mean of at least three independent experiments. * p < 0.05. (G) Transwell system is used to allow calcein-labeled neutrophils to cross an endothelial monolayer that was cultured on a FN-coated filter with 3 µm pore size including fluorescent block. Increase in fluorescence was detected in real-time using a fluorimeter and showed increased neutrophil migration towards C5a. Squares represent shCTRL-ECs and open circles represent shADAM10-ECs. Graph on the right shows quantification after 20 min. Data are mean of at least three independent experiments. * p < 0.05.