In Vitro Internalization Studies

For internalization experiments, MDA-MB-231 cells were plated in six-well plates at a density of 600 000 cells/well and allowed to recover for 24 h at 5% CO₂ at 37 °C. On the day of the assay, the cells were incubated with approximately 0.74 MBq/well of [¹⁸F]MCFB for 30 min at 4 °C (assuming inhibition of receptor internalization at this temperature) or at 37 °C. Under conditions incubated at 4 °C, the media was replaced with fresh 37 °C media and incubated for 5, 15, 30, and 60 min at 37 °C. After incubation, surface-bound radioactivity was removed by washing the cells with 400 μL of ice-cold 50 mM glycine in 150 mM NaCl (pH 3) for 5 min on ice, followed by two washes with ice-cold PBS. To obtain the internalized fraction, the cells were lysed with 400 μL of 1 M NaOH. The radioactivity of the internalized fraction was measured by γ-counting. Counts/min data were expressed as a percentage of the ID of radioactivity in each well and the total cellular protein concentration of the sample, determined using the Pierce BCA assay, that is, % ID/mg protein. The percentage of internalization was calculated as