Antibodies against CD99 and CD99L2 do not interfere with neutrophil lateral migration on endothelioma cells but selectively inhibit neutrophil transendothelial migration in vitro. bEnd.5 cells were cultured to confluence on laminin-coated coverslips and were incubated with 30 μg/mL of the indicated antibody for 15 minutes before the assay. Neutrophils were added on top of the bEnd.5 cell monolayer and were allowed to adhere for 10 minutes. Lateral migration (A-C) and transmigration of neutrophils (D) across the endothelial cell monolayer were visualized by time-lapse video microscopy. Phase-contrast images were captured every 20 seconds over a time period of 20 minutes. (A) Cell tracks of 4 individual neutrophils on a bEnd.5 cell monolayer in the presence of co-IgG. (B) Tracking data of 6 individual neutrophils (of 70 evaluated) for each antibody investigated. (C) Quantitative analysis of the distance that neutrophils migrated on endothelial cells during the 20-minute period in the presence of the indicated antibodies. (D) Percentage of transmigration of neutrophils across bEnd.5 monolayers during the time interval. ***P < .001. **P < .01. Statistical analysis was done by ANOVA evaluating all available data.