Neurotensin

The protocol and recommendations edited by Reubi and co-workers for binding assays were strictly adhered to [20 (link)]. Frozen samples were kept at −80 °C. Three days before handling, samples were placed at −20 °C. The day of the experiment, samples were pre-incubated for 10 min at 37 °C in Tris-HCl buffer at pH 7.4. Then, a binding solution containing 10 nM of the radiopharmaceuticals (except [¹¹¹In]In-JMV 7488 and [⁶⁷Ga]Ga-pentixafor, which were used at 75 nM and 50 nM, respectively) in Tris-HCl buffer at pH 8.2, 1% of BSA (Sigma A2153), 40 μg/mL of bacitracin (Sigma^®11,702), and 10 nM of MgCl₂ (Sigma M8266) was applied. In order to determine the amount of non-specific binding, a large excess of cold ligand was added—more precisely, 1μM of [^natGa]Ga-RM2 (Life Molecular Imaging), [^natGa]Ga-PSMA-617 (ABX), neurotensin (Bachem), or [^natLu]Lu-DOTATATE (ABX), 7.5 µM of levocabastine or 10 µM pentixafor were used. Samples were incubated at 37 °C for 2 h. Afterward, samples were rinsed five times for 8 min in cold Tris-HCl buffer at pH 8.2 with 0.25% of BSA, two times for 8 min in cold Tris-HCl buffer at pH 8.2 without BSA and finally, two times for 5 min in distilled water.