2480 wizard2 gamma counter

Isolated splenic B cells were incubated for 3 hours at 37°C with protamine-conjugated ⁸⁹Zr Feraheme nanoparticles^{14 (link)} at a concentration of 100 µg/ml iron. This incubation time was previously determined to be sufficient for labeling the majority of the B cells in suspension (data not shown). To remove unbound nanoparticle, the cells were centrifuged for 5 minutes at 500×g and re-suspended in fresh PBS three times, after which the cells were finally re-suspended in PBS at a concentration of 100,000 cells/µl. Approximately 2 million B cells in 20 µl of PBS, comprising 20 µCi of total radioactivity were applied the wound on the right dorsal side of the animal as described above, while the contralateral wound received an equal volume of PBS. The mice were imaged at regular intervals using an eXplore Vista micro-PET-CT imaging system (GE Healthcare). At the end point of the studies, the animals were euthanized and tissue samples from the treated wound site, the control wound, lymph nodes on each side of the body, the spleen, liver and muscle were collected for measuring residual radioactivity using a 2480 WIZARD² gamma counter (Perkin Elmer).

Plasma Free Fraction (f_P) was measured using rapid equilibrium dialysis (RED). Plasma samples were isolated by centrifugation from one blood sample obtained from each participant before radiotracer injection. A 300 μL sample of plasma was spiked with 3 μCi of [¹⁸F]ASEM and added into the sample chamber of Single-Use RED Plate with Inserts (Thermo Scientific, Rockford, IL, USA) with an 8 K molecular-weight cutoff. 500 μL of PBS was added to the buffer chamber and the plate was incubated on an Incubating Microplate Shaker (Fisher Scientific, Waltham, MA, USA) at 37°C for 4 h. 100 μL of plasma and 100 μL of PBS were transferred to a clean tube. All samples were measured in triplicate. The radioactivity was measured using a 2480 WIZARD² Gamma Counter (Perkin Elmer, Waltham, MA, USA) to obtain the radioactivity in the plasma (C_P) and buffer (C_U). The free, unbound fraction (f_P) was calculated as: f_P = C_U / C_P × 100 (%).

The competitive cell binding assay was used to assess the in vitro NTR1 binding affinity and specificity of NT-IRDye800 and NT-AmBaSar based on the previous reported method (Alshoukr et al. 2011 (link); Wu et al. 2014 ). In brief, 50% inhibitory concentration (IC₅₀) for the binding to living HT29 cells was determined by competitive receptor binding between 125I-labeled neurotensin (Perkin-Elmer) and non-radiolabeled NT analogs. HT29 cells (2 × 10⁶ cells) were rinsed with 500 μL DMEM supplemented with 0.2% BSA and incubated for 60 min at 37°C with ¹²⁵I-neurotensin (0.25 uCi, 300 μL DMEM, 0.2% BSA, 0.8 mM 1, 10-phenanthroline) in the presence of increasing concentrations of non-radiolabeled IRDye800-NT, AmBaSar-NT, or unmodified NT peptide. After being washed twice with ice-cold DMEM 0.2% BSA, cells were lysed in 1 mL 2 N NaOH and radioactivity was counted using 2480 WIZARD² gamma counter (Perkin Elmer). The data were fitted with nonlinear regression using GraphPad Prism (GraphPad Software) to calculate the 50% inhibitory concentration (IC₅₀) value. Experiments were performed in triplicate.