ABSTRACT

HEK purchased as second-passage cells were maintained in KGM at 37°C under 5% C02 and subcultured as previously described by Smith et al.10 Third-passage cells were plated at 20,000-30,000 HEK/well onto 25-mm Falcon tissue culture inserts with a pore size of 0.45 mm (Becton-Dickinson & Co., Lincoln Park, NJ). The supplier’s documents indicated these inserts had been precoated with 5|xg/cm2 human plasma fibronectin (Sigma Chemical Co., St. Louis, MO). Cell counts and viabilities were determined on HEK harvested with trypsin-EDTA.10 Cells were either counted manually using a hemacytometer or in a Coulter counter ZM (Coulter Electronics, Hialeah, FL). Viabilities were determined by propidium iodide dye exclusion using a Coulter EPICS C flow cytometer.10