Measurement of cell uptake of radioactive ligands

Cells were seeded in 6-well plates (Sigma-Aldrich) until they reached approximately 80% confluency. Before the uptake experiments, the cells were preincubated with glucose-free RPMI-1640 medium (Gibco) for 4 h. The cells were then trypsinized (Gibco), counted, and 1×10⁵ cells transferred into a 5 mL test tube containing warmed Hank's balanced salt solution (HBSS, Sigma-Aldrich) with 0.5% (w/v) bovine serum albumin (Sigma-Aldrich). Subsequently, approximately 0.185 MBq of radioactive ligands were added to the tubes, and the cells were incubated in a humidified incubator with 5% CO₂ for 1 h at 37°C. The cells were then washed 3 times with cold HBSS and lysed for 5 min in 200 μL of 1% sodium dodecyl sulfate (SDS, Sigma-Aldrich). Cell lysates were collected, and radioactivity was measured by a Cobra II gamma counter (Canberra Packard; Vaughan, Ontario, Canada). Radioactivity was normalized according to the amount of total protein at the time of assay. Total protein levels were quantified by the BCA protein assay kit (Pierce, Rockford, IL, USA). All experiments were performed in triplicate.