kinetics and movement of monocyte-derived dendritic cells across the endothelium responding to CCL2. (A) Human brain microvascular cells (hCMEC/D3) were grown to confluence on collagen-coated polyethylene tetraphthalte membrane transwell inserts with 3 μm pores. Monocyte-derived dendritic cells (MDDCs) (2×105) were labeled with 4',6-diamidino-2-phenylindole (DAPI) and added to the upper chamber while medium with or without 100 ng/ml CCL2 was added to the lower chamber. At indicated times, transwells were washed, stained for ICAM-1 (green) or caveolin-1 (red) and viewed at 20× magnification. (B) MDDCs and peripheral blood lymphocyte (PBLs) were labeled with DAPI and added to the upper chamber of the transwell. At 30 minutes after addition of the immune cells, the transwells were washed, stained for ICAM-1 (green) or caveolin-1 (red) and viewed at 100× magnification to determine paracellular versus transcellular migration patterns. (See also Additional file
1: Movie S1 and Additional file
2: Movie S2.).