Optiquant software

To evaluate the time course of radiolabeling, 45 µL of Fe₃O₄@Al(OH)₃ NPs containing 60 µg of Fe were incubated with 5–30 MBq [¹⁸F]NaF (5–30 µL) while shaking. Two, five, and 10 min after labeling, 2 µL samples were taken and blotted on instant thin layer chromatography (iTLC) papers impregnated with silica gel (iTLC-SG papers; Varian, Diegem, Belgium). The papers were developed in an elution chamber using NaCl 0.9% as the mobile phase. The read-out was performed using a 2480 Wizard² Automatic Gamma Counter (20 s protocol; PerkinElmer, Waltham, MA, USA) after splitting the papers into two equal halves representing the unbound and bound radiotracer. In addition, autoradiography was performed using phosphor screens (Perkin Elmer) in standard film cassettes. The screens were removed from the cassettes after a five-minute exposure and were scanned immediately at 300 dpi resolution using a Cyclone Plus System (Perkin Elmer). Images were analyzed using the manufactures’ Optiquant software (version 5; Perkin Elmer).
For further in vitro and in vivo experiment, NPs are labeled with [¹⁸F]NaF for ten minutes in Milli-Q water. Afterward, they are centrifuged for 20 min at 4000 rpm and resuspended in the media of choice for further application.

Fractions of blood collected during biodistribution studies were used to assess metabolic stability of [¹⁸F]FPyPEGCBT-c(RGDfK). Metabolic activity was quenched (v/v) with a solution of 20 % MeCN in PBS (100 mM, pH 7.4). Blood samples were centrifuged at 4 °C (2000 × g, 8 min) and the serum was removed. Serum proteins were precipitated with MeCN (1:1) and centrifuged at 4 °C again (21380 × g, 8 min). Supernatants were removed from pellets, and the activity associated with both was measured. In this fashion, the extraction efficiency of this step was estimated to be 91.0 % ± 4.8 (n = 18). For each sample, an aliquot of supernatant (2 μL) was spotted onto silica gel plates and eluted with 4:6 MeOH-10 % (w/w) aqueous ammonium acetate. Phosphor imaging screens were exposed to dried TLC plates for 10–25 min and analysed by autoradiography using a Cyclone plus and the built-in Optiquant software (Perkin Elmer).

Resection of DSB ends was analyzed at an HO endonuclease-induced DSB at the MAT locus on chromosome III using Southern blots as previously described (7 (link),11 (link)). Galactose induction, sample collection, DNA isolation and purification were carried out as described by Chen et al. (11 (link)). Purified DNA was digested with EcoRI and separated on a 0.8% agarose gel followed by transferring onto a nylon membrane. Radiolabeling of DNA probes was performed following manufacturer's instruction (Takara). Southern blotting and hybridization with radiolabeled DNA probes was performed as described previously (7 (link),11 (link)). The blot was exposed in a Phosphor screen. Signal on the screen was captured by scanning in an OptiQuant Cyclone Plus machine (Perkin Elmer). Intensities of target bands were analyzed with OptiQuant software (Perkin Elmer) and normalized to the TRA1 probe. Resection rate was calculated as previously described (7 (link)). Three independent experiments were performed for each strain.