Induction of reduced endothelial permeability to horseradish peroxidase by factor(s) of human astrocytes and bladder carcinoma cells: detection in multi-well plate culture.

Endothelial permeability to horseradish peroxidase (HRP) was assayed in multi-well plate culture. Confluent cobblestone-monolayers of endothelial cells were incubated with HRP, and the amounts of HRP that permeated the monolayer and reached the bottom substrate were estimated by extraction of HRP with deoxycholate and following addition of chromogenic substrate of HRP into the extracts. Using this in situ assay method, we detected the reduced permeability to HRP in aortic and brain microvascular endothelial cells after 4-5 days culture with conditioned media of human astrocytes or bladder carcinoma cells. Preliminary characterization of the active factors(s) released from these cells was performed. This method will be useful for monitoring impermeable endothelial cell monolayers and identifying the active factor(s).