2480 automatic gamma counter wizard2 3

Binding to and internalization of [⁶⁸Ga]Ga-PSMA-11 into LNCaP cells were assessed in vitro as previously described [40 (link)]. Briefly, an aliquot containing 2 × 10⁶ LNCaP cells in RPMI-1640 medium (450 µL) was added to a vial and incubated with [⁶⁸Ga]Ga-PSMA-11 (50 µL/~1 MBq) at 37 °C and under slight agitation for 30 and 60 min (n = 5 for each time interval). Then, the vials were centrifuged (825 g, 5 min) with Minispin^® plus (EppendorfAG—Hamburg, Germany). The cell pellets and supernatants were separated, and their radioactivities were measured in a Wizard²™ 3” 2480 automatic gamma counter (PerkinElmer—Norwalk, CT, USA). Binding to LNCaP cells was calculated as follows (Equation (4)): $\mathrm{Binding}=\frac{\mathrm{cpm} \left(\mathrm{pellet}\right)}{\mathrm{cpm} (\mathrm{pellet}+\mathrm{supernatant})}\times 100\%$
After that, pellets were resuspended in 0.5 mL of acid wash buffer (0.2 M acetic acid in 0.5 M NaCl solution, pH 2.8) and kept at room temperature for 5 min to remove cell-surface-bound [⁶⁸Ga]Ga-PSMA-11. Then, the vials were centrifuged (825 g, 5 min) with Minispin^® plus (EppendorfAG—Hamburg, Germany). The cell pellets and supernatants were separated and their radioactivities were measured in a Wizard²™ 3” 2480 automatic gamma counter (PerkinElmer—Norwalk, CT, USA). Internalization into LNCaP cells was calculated as follows (Equation (5)): $\mathrm{Internalization}=\frac{\mathrm{cpm} \left(\mathrm{pellet}\right)}{\mathrm{cpm} (\mathrm{pellet}+\mathrm{supernatant})}\times 100\%$

3 × 10⁵ cells were seeded into each well of a 24 well plate in their appropriate medium containing 0.5% FBS and the inhibitors and concentrations indicated. After incubation for 24 h, cells were harvested by centrifugation and resuspended in glucose-free RPMI medium (Thermo Fisher) supplemented with 5.5 mM glucose [25 (link)] and 0.5% FBS and the inhibitors indicated. After 30 min, 100 kBq of FDG was added to the cells and samples were incubated for the indicated times or for 30 min at 37 °C. The cell suspension was pipetted onto a Costar SpinX centrifuge tube filter column (0.45 µm, Corning, Corning, NY, USA) and centrifuged for 1 min at 4 °C to retain the cells with the incorporated radioactivity. The flow through was discarded and cells were washed two times with cold PBS. Cell-bound radioactivity was measured with a gamma counter (2480 Automatic Gamma Counter Wizard² 3, Perkin Elmer, Waltham, MA, USA). A parallel plate was treated in the same way and cells were harvested and lysed in cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) for. The lysates were centrifuged at 10,000×g for 10 min at 4 °C and the protein concentrations of supernatants were determined with a modified Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA).