Pulse Chase Assay of rhTF in Caco-2 and HeLa Cells. Confluent Caco-2 or HeLa cells were pulsed for 1 hr by incubation with pulse medium containing 3 μg/ml 125I-labelled hTF or rhTF at 37°C. Then, the pulse medium with radiolabeled TF was removed by aspiration of the medium followed by washing cell monolayers with cold DMEM medium supplemented with 0.1% BSA. Caco-2 and HeLa cells were then chased by incubation with excess molar (0.3 mg/ml) of unlabeled hTF or rhTF in chase medium at 37°C. After chasing for 1 or 3 hr, the percentage of cell-associated (A) and released (B) hTF or rhTF was determined. Data is represented as average with error bars indicating standard deviation, n = 3.