Damage caused by the test compound to the tight junctions in MDCK monolayers is determinedby the amount of fluorescein which leaks through the cell layer and is an indication of potential tocause eye irritancy.
MDCK cells are maintained in culture and exposed for a short period, suchas one minute, to five fixed concentrations of test compound. The amountof damage caused to the tight junctions is determined by the amount offluorescein which leaks through the cell layer over a set period of time. Thisis compared to two controls: the amount of fluorescein which leaks throughan untreated confluent monolayer, referred to as 0% leakage or the blank;and the amount of fluorescein which leaks through an insert on which thereare no cells, referred to as maximum leakage. The percentage of leakageand therefore amount of damage to the tight junctions is expressed,relative to these controls, for each of the set concentrations.