Figure 1. Primary monocytes lose their ability to complete transendothelial migration after 16 h ex vivo incubation at 37°C under static conditions.

Freshly isolated human monocytes (left column; Supplemental Video 1) or monocytes incubated for 16 h at 37°C in a polypropylene tube (right column; Supplemental Video 2) were added to TNF-α-activated HUVEC monolayers, and monocyte transendothelial migration under static conditions was monitored for 2 h by time-lapse video microscopy. The ex vivo incubation time was selected to allow optimal knockdown of proteins of interest in primary monocytes using siRNA constructs [24]. Individual monocytes were tracked on the apical surfaces up to the last frame or completion of diapedesis, which was defined as the frame where the monocyte cell bodies, retracting between endothelial junctions, disappeared from the apical surface of HUVEC monolayers. A monocyte that underwent diapedesis was evaluated as one that completed transendothelial migration, and the data are expressed as percent of total monocytes in the microscopic field over a 2-h observation period. Means ± sem from independent experiments using three different monocyte donors are shown; *P < 0.01 (paired t-test).