Wizard 3

The positron-emitting starting reagents [¹²⁴I]NaI (t_1/2 = 4.18 days) and [⁸⁹Zr]Zr-oxalate (t_1/2 = 3.17 days) were provided by the Memorial Sloan Kettering Radiochemistry & Molecular Imaging Probes Core Facility. Activity measurements were made either with a dose calibrator (Capintec) or an automated gamma counter (PerkinElmer Wizard 3). For ⁸⁹Zr radiolabeling, J591 was conjugated with isothiocyanatobenzyl-desferrioxamine (SCN-DFO; Macrocyclics, Dallas, TX) and subsequently radiolabeled with ⁸⁹Zr-oxalate as described [9 ] (specific activity (SA) 118 MBq/mg; radiolabeling efficiency (RE) 70 %; minimum immunoreactivity (IR) 90 %). J591 was radiolabeled with ¹²⁴I as previously described [24 (link)] using the IODOGEN method to a SA of 213 MBq/mg (RE 60 %; IR 75 %). The radiochemical purity of each tracer was determined using either instant thin-layer chromatography with 5 mM DTPA pH 5.0 or 10 % trichloroacetic acid as the elution solvent for ⁸⁹Zr-mAb or ¹²⁴I-mAb, respectively, and was routinely >98 %. To prepare the different mAb mass doses, non-radioactive J591 was added as necessary to achieve the final desired mAb mass after drawing up the ⁸⁹Zr-J591 [155-243 μCi (5.7–9.0 MBq) per animal] or ¹²⁴I-J591 [171-267 μCi (6.3–9.9 MBq) per animal], based on the initial SA of the respective preparations.

Log D ratios of ⁶⁴Cu^II‐labelled bispidine ligands 2–6 were determined using 1‐octanol/buffer mixtures. The experiments were performed with 100 μm solutions of bispidine ligands dissolved in aqueous buffer solutions. Aqueous phases consisted of 440 μL of 50 mm 4‐(2‐hydroxyethyl)‐1‐piperazine ethanesulfonic acid (HEPES)‐NaOH buffer (pH 7.2, 7.4, 7.6), 10 μL of [⁶⁴Cu]CuCl₂ solution (500 kBq) and 50 μL of a 100 μm Cu(NO₃)₂ solution. The distribution experiments were carried out at 25±1 °C in microcentrifuge tubes (2 cm³) with mechanical shaking for 30 min. The phase ratio V(_1‐octanol):V(_aq) was 1:1 (0.5 mL each). Full complexation was checked by radio‐TLC, which gave no evidence of free copper(II) in the aqueous phase. All samples were centrifuged and the phases then separated. The copper(II) complex concentration in both phases was determined radiometrically using γ‐radiation (⁶⁴Cu, NaI(Tl) scintillation counter automatic gamma counter 1480, Wizard 3′′, Perkin–Elmer). The results are the average values of three independent experiments.

The radioligand
binding assay was performed using [¹²⁵I]CLINDE as the radioactive
TSPO ligand for evaluation of the density in TSPO. [¹²⁵I]CLINDE synthesis protocol can be found here,^{26 (link)} but briefly, the CLINDE tributyltin precursor was incubated
in acetic acid with Na¹²⁵I (PerkinElmer) and peracetic
acid before purification using a reversed-phase column.
C6 cells
were seeded at 1.0 × 10⁵ cells/well density in 24-well
plates. After 8 h of incubation at 37 °C, 5% CO₂ medium
was replaced with fresh medium containing [¹²⁵I]CLINDE
(at 10 different concentrations from 0.005 to 1 μCi/well). The
medium was supplemented with LPS from Escherichia coli (10 μg/mL, Sigma-Aldrich, L2630) for the treated group and
with FEPPA (N-(2-(2-fluoroethoxy)benzyl)-N-(4-phenoxypyridin-3-yl)acetamide, 10 μM, ABX advanced
biochemical compounds, Germany) for the determination of the nonspecific
binding. After overnight incubation at 37 °C, 5% CO₂ cells were washed three times with 50 mM Tris HCl and 50 mM MgCl₂ buffer. The cells were detached using 300 μL/well of
triple detergent buffer (Tris 1 M pH 8, NaCl, azide sodium 10%, SDS20%,
NP40 (IGEPAL CA-630), deoxycholate sodium (D6750-10G), supplemented
with inhibitors of proteases and phosphatases) and immediately counted
using a γ counter (Wizard 3, PerkinElmer).