FIG. 1.

Human BMEC in vitro express P-glycoprotein and tight junctional proteins. (A) Confluent and subconfluent human BMEC monolayers were lifted with EDTA. The cells were fixed with 2% paraformaldehyde in PBS for 30 min, permeabilized with PBS containing 0.01% Triton X-100, and quenched with 10 mM glycine-PBS. After blocking with 10% normal goat serum, the cells were incubated with fluorescein isothiocyanate-labeled mouse anti-P-glycoprotein monoclonal antibody and rabbit anti-factor VIII-Rag polyclonal antibody, followed by secondary antibody coupled to phycoerythrin. Analysis was done by using a FACScan and the CellQuest software (Becton Dickinson, San Jose, Calif.). The percentages of gated human BMEC positive for both factor VIII-Rag and P-glycoprotein are shown. (B) Human BMEC monolayers were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained with anti-human ZO-1 monoclonal antibody. The presence of ZO-1 was visualized with Alexa 488-conjugated secondary antibody. Differential interference contrast (left panel) and fluorescence (right panel) images of human BMEC showed that ZO-1 is expressed at the cell junctions. Magnification, ×400.