Quantification of cellular internalization

Human breast cancer cell line MCF7 and human T cell line Jurkat were seeded in 24 well plates (1x10⁵ cells/well). Cells were treated with different concentrations of FITC-labelled peptides for different periods of time. After the treatment with FITC-labelled shuttles, cells were harvested and washed twice with PBS to remove the extracellular unbound peptide. Cells were treated with trypsin for 5 min to remove non-internalized surface bound peptide and then, centrifuged, washed and resuspended in 200 ml of PBS. FITC fluorescence intensity of internalized peptide was measured by flow cytometry (BD Biosciences) by acquiring 10,000 live cells. Experiments were carried out three times in duplicate. Untreated cells were used as control.