Cyclone plus phosphor imager

Pulse-chase labeling of B. subtilis was performed with 25 μCi Easy tag [³⁵S]methionine (PerkinElmer) as previously described (23 (link)). Subsequently, ³⁵S-labeled AmyQ was immunoprecipitated with protein A affinity medium (Mabselect Sure; GE Healthcare Life Sciences) and specific polyclonal antibodies. Precursor and mature forms of the immunoprecipitated labeled AmyQ were separated by lithium dodecyl sulfate-PAGE using 10% NuPage gels (Life Technologies) and visualized using a Cyclone Plus PhosphorImager (PerkinElmer) as previously described (23 (link)).

In short, following a previously published protocol [12 (link),19 (link),20 (link)], 100 µL of sterile sodium acetate solution (0.4 M, pH 5.5) and 1.25 µL ascorbic acid solution (20% w/w) were added to ca 30–40 µL (1.25 GBq in total) of non-carrier added Lutetium-177 (ITM Isotope Technologies Munich, Garching, Germany). To this solution, 20 nmol (volume circa 2 µL) of PSMA-617 (MedChemExpress, Monmouth Junction, NJ, USA) were added and labeled by incubating on a shaker at 95 °C for 15 min after which the reaction was terminated by cooling to room temperature. At 0 and 15 min, respectively, 1 µL of the solution was added to an instant thin layer chromatography (iTLC) strip. Sodium citrate solution (0.2 M, pH 2) was used as mobile phase, and the percentage of free ¹⁷⁷Lu (which migrates with the solvent front) was determined by analyzing the iTLC strips with a phosphor imager system (Cyclone Plus Phosphor Imager, PerkinElmer, Inc., Waltham, MA, USA). A sterile 0.9% sodium chloride solution was added, and the radioligand diluted (1:3 or 1:7), a sample for iTLC taken, and pH tested before injections in mice. ¹⁷⁷Lu-PSMA was labeled at a specific activity of 62 MBq/nmol (judged most effective for therapy by Fendler et al. [19 (link)]), and the radiochemical purity of the radioligand was >99%.

For mobility information, soil thin layer chromatography was carried out (Soil-TLC), according to the US Environmental Protection Agency OPPTS 835.1210 [65 ]. The soil samples from different soil systems (NC, SC-NT, SC-CT, and SG-MC) and deep soil + fresh organic material strips in a standard soil were used in the soil-TCL plates (9 cm wide, 15 cm long, and 2 cm thick). The plates were made in duplicate, and ¹⁴C-metribuzin from MTZ commercial formulation and nanoMTZ formulation was applied (833.33 Bq) in each plate, to visualize the herbicide mobility. The plates were gently placed in chromatographic chambers containing 100 mL of deionized water until the elution limit line (10 cm above application points) was complete. Subsequently, all plates were dried at room temperature for 48 h. Autoradiograph images were obtained using phosphorescent films, read in radio scanner equipment (Cyclone Plus Phosphor Imager, Model C431200, PerkinElmer Inc., Shelton, CT, USA). Rf (retention factor) values were calculated based on the distance traveled by the herbicide on the plate, using Equation (4):
where Dh is the distance from baseline (application point) traveled by herbicide on the plate and Ds is the distance from baseline (application point) traveled by the solvent (water) on the plate (in this case, 10 cm).