Hydrochloric acid (hcl)

Ten micrograms of DTPA-cNGR was dissolved in 0.5 ml of NaAc buffer (0.5 M, pH6) and radiolabeled with ¹¹¹InCl₃ (400 to 600 MBq, in ± 0.2 ml 0.1 M HCl, Mallinckrodt, Petten, The Netherlands) at room temperature for 30 min. Product purity was checked by HPLC (Shimadzu Corporation, Columbia, MD, USA) using an Aeris WIDEPORE 3.6u XB-C18 column 250 × 4.6 mm (Phenomenex, Utrecht, The Netherlands) eluted with a gradient from 0.1% trifluoroacetic acid (TFA) in water (0 to 3 min) to acetonitrile (ACN) over the course of 20 min at a flow rate of 1 ml/min.

DNA synthesis and cell cycle distribution were assayed by flow cytometry as previously described with some modifications [13 (link),44 (link)]. The ethanol-fixed tumor tissue was washed in cold PBS and incubated in 1 mL 0.04% pepsin (w/v; Sigma) in 0.1 N HCl (Mallinckrodt, Inc., St Louis, MO, USA). After 1 h at 37 °C with shaking, the digest was centrifuged and the pellet re-suspended in 2 N HCl (1 mL; Mallinckrodt, Inc.), and then incubated at 37 °C for 20 min with shaking. The nuclear suspension was neutralized with 0.1 M sodium tetraborate (Sigma), washed with phosphate buffered saline containing 0.5% bovine serum albumin and 0.5% Tween-20 (PBTB) and re-suspended in PBTB. The nuclei (100 µL) were incubated with 20 µL of FITC-labeled anti-BrdU mAb (BD Biosciences, San Jose, CA, USA) for 1 h at 4 °C, washed in cold PBS and re-suspended in 2 mL of propidium iodide solution (50 µg/mL in PBTB; Sigma) containing RNAse A (50 µg; Sigma) and incubated for 30 min at 4 °C. Flow cytometry was performed using a FACSCalibur (BD Biosciences), collecting 20,000 events. The DNA content (propidium iodide) and DNA synthesis (BrdU content) were analyzed using two parameter data collection using CellQuest (BD Biosciences) software. ModFit LT software (Verity Software House, Topsham, ME, USA) was employed to perform a single parameter analysis of the cell cycle distribution.

The reagents used to pretreat and impregnate the raw materials were sulphuric acid (98%) purchased from Merck (Darmstadt, Germany) and phosphoric acid (85%) from Mallinckrodt (Utrecht, The Netherlands). The chemicals and reagents used for preparing metal ion solutions, modifying carbon and detecting Cr(VI) were Ni(II) nitrate hexahydrate (Fluka), lead (II) nitrate (Riedel-deHaën), and potassium dichromate (Merck ), nitric acid (65%, Merck), hydrochloric acid (37%, Mallinckrodt), sodium hydroxide pellets (Merck), iron (III) chloride-6-hydrate (Hamburg Chemicals), calcium (II) hydroxide (GCE), sodium chloride (Merck), 1,5-diphenylcarbazide (Merck) and acetone. All chemicals and reagents were of analytical grade.

Modification of anti-claudin-4 mAb with p-SCN-Bn-DTPA and subsequent radiolabelling with indium-111 were conducted following methods described by Brom et al. [16 ]. In brief, to a solution of anti-claudin-4 MAB4219 (200 μg) or mouse IgG_2A (200 μg, MAB003, R&D Systems) in 0.1 M NaHCO₃ (pH 9, 100 μl, Chelex treated) was added 20 M equivalents of p-SCN-Bn-DTPA (245.6 μM) in anhydrous dimethyl sulphoxide. The reaction mixture was incubated at room temperature for 30 min with gentle shaking (450 rpm), and the excess p-SCN-Bn-DTPA was removed by Sephadex-G50 size exclusion chromatography (Sigma-Aldrich). The affinity of the DTPA-conjugated MAB4219 for claudin-4 was evaluated by flow cytometry in Panc-1 and HT1080 cells and compared to that of the unmodified antibody.
Indium-111 in 0.02 M hydrochloric acid (sourced from Mallinckrodt Pharmaceuticals) was added to a 2-mg/ml solution of the DTPA-modified antibody to achieve a ratio of at least 1 MBq to 1 μg. The reaction mixtures were incubated at room temperature for 1 h, and the radiolabelling efficiency was determined by iTLC using an eluent of 0.1 M sodium citrate buffer (pH 5.5). The crude reaction mixture was purified by Sephadex-G50 size exclusion chromatography, eluting with 100-μl fractions of phosphate-buffered saline (pH 7.4).