Cyclone storage phosphor system

Radioiodination of scFvD2B with ¹²⁴I (half-life: 4.176 days) was performed as following: Na ¹²⁴I (Perkin Elmer), after oxidation in an iodogen-coated tube (Pierce, Rockford, IL) with 0.1 ml of iodination buffer (TRIS-HCl 25 mM, pH 7.4 + NaCl 0.4 M) for 5 min at room temperature, was added to scFvD2B for 10 min at room temperature with gentle rotation.
The radiolabeled reagent was purified using a PD-10 desalting column and eluted with Sodium Phosphate buffer 100 mM pH 7.4 + NaCl 150 mM + Ascorbic Acid 10 mg/ml. The fractions corresponding to the radiotracer peaks were pooled and counted into a COBRA II Auto-Gamma counter (Packard, PerkinElmer, Boston, MA). The labelling efficiency before gel filtration and the final radiochemical purity after purification, calculated as amount of radioactivity associated to the protein vs the fraction of free iodine, available after the reaction, were determined by instant thin-layer chromatography on silica gel strips (ITLC-SG; Gelman Sciences, Ann Arbor, MI) using Methanol/Physiologic Sodium Chloride solution (1:1, v/v), as the mobile phase, read by Cyclone Storage Phosphor System and analyzed by Optiquant Image Analysis Software (Packard).

Indium chloride [¹¹¹In]InCl₃ was purchased from Mallinckrodt Pharmaceuticals (Staines-upon-Thames, UK). Sodium iodide [¹²⁵I]NaI was purchased from Perkin Elmer Sverige AB (Hägersten, Sweden). Instant thin-layer chromatography (iTLC) analysis was performed using iTLC silica gel strips (Varian, Lake Forest, CA, USA). The activity distribution was measured using a Cyclone storage phosphor system (Packard) and analyzed by OptiQuant image analysis software (Perkin Elmer, Waltham, MA, USA). Purification was performed using NAP-5 columns (GE Healthcare, Little Chalfont, United Kingdom) pre-equilibrated with 1% BSA in PBS and eluted with PBS. Activity was measured using an automated gamma-spectrometer with a NaI(TI) detector (1480 Wizard, Wallac, Finland). BxPC-3 and DU145 cells were purchased from the American Type Culture Collection (ATCC) and were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/mL penicillin, and 100 µg/mL streptomycin in a humidified incubator with 5% CO₂ at 37 °C, unless stated otherwise.

Total RNA from Anabaena was isolated as described in reference 69 (link), and trace DNA in the samples was removed by treatment with Turbo RNase (Ambion) following the manufacturer’s instructions. Northern blot assays were performed as described in reference 70 (link), with 4 μg RNA loaded per lane, and electrophoresed in denaturing 1% agarose formaldehyde gels. DNA probes were internal gene fragments generated by PCR using Anabaena genomic DNA and primer pairs all0087-22/all0087-23 (mreB), all0086-13/all0086-14 (mreC), and all0085-8/all0085-9 (mreD). The rpnB gene, which was used for normalization, was amplified from plasmid pT7-7120 (71 (link)) with the primers Universal and Reverse. Probes were labeled by annealing the PCR-generated fragments to oligonucleotides complementary to the coding strand (all0087-8/all0087-23 for mreB, all0086-6/all0086-14 for mreC, and all0085-5/all0085-9 for mreD) and polymerization catalyzed by the Klenow fragment of DNA polymerase (Thermo Fisher) in the presence of [α-³²P]dCTP (Perking-Elmer). Radioactive areas in Northern blot hybridization membranes were visualized and quantified with a Cyclone storage phosphor system (Packard).