4.4.3. Internalization and Cell Uptake Assay

Cells were seeded (1.2 × 10⁶ cells/well) and cultured to confluency for 24 h prior to experiments. We prepared 10⁻⁹ M solutions of [¹¹¹In]In-PSMA-617, [¹¹¹In]In-22, and [¹¹¹In]In-30 in internalization media (RPMI, 20 mM HEPES, 1% BSA, pH 7.4). The media were removed from each well, and they were rinsed twice with PBS at rt. 2 mL of the radioactive compound was then added to each well and incubated at 37 °C for 2 h. For the internalization assay, the media containing radioactive compound were removed, and each well was washed twice with cold PBS. A total of 1 mL of Glycine buffer (50 mM glycine, 100 mM NaCl, pH 2.8) was added immediately and incubated for 10 min at rt. The glycine wash was then collected into counting tubes (membrane-bound fraction). An extra wash was performed and collected in the same tubes. A total of 1 mL of NaOH (1 M) was then added to each tube and left for 15 min at rt. The lysate (internalized fraction) was then collected in separate counting tubes, together with one extra NaOH wash. Data were normalized to 1,000,000 cells and percentage added dose.