Basal-to-apical transendothelial migration of MP. (A) Unstimulated endothelial cells grown on collagen were incubated with PBMC for 2 hours, then rinsed to remove nonadherent cells. Some cultures were then processed for analysis; others were incubated further for as long as 7 days. Two methods were used to analyze reverse transmigration experiments: visual enumeration of MP beneath endothelial monolayers using Nomarski interference optics (filled squares) and assessment of MP content in cultures using the DNA-binding dye Yo-Pro-1 (filled diamonds). Percent reverse transmigration is defined as the percentage decrease in the number of MP beneath the endothelium, relative to the number of subendothelial MP at 2 hours. (B) Some cultures were prepared that contained FITC-conjugated beads embedded in the collagen. Using flow cytometry, the uptake of these beads by MP that migrated into the collagen was assessed at 24 hours, after the cells were removed from the collagen with collagenase, and compared to the extent of beads associated with MP that accumulated in the apical compartment of parallel cultures between 24 and 48 hours of incubation. Profiles represent histograms from a representative experiment of MP collected from fluorescent bead–containing cultures (shaded) or MP from cultures without beads (unshaded).