Zr/In-oxine labeling of WBCs for γH2AX analysis

WBC labelled with [⁸⁹Zr]Zr-oxine or [¹¹¹In]In-oxine or treated with PBS only were suspended in assay medium, seeded onto poly-L-lysine-coated coverslips and incubated for 30 min at 37 °C. Fixation and staining for γH2AX were performed as previously described [35 (link)]. Briefly, the cells were fixed and permeabilised with 3.7% formalin, 0.5% Triton X-100 and 0.5% IGEPAL® CA-630 in PBS. Staining was performed with an anti-γH2AX (Ser139) mouse mAb (1:1600; JBW301, Merck #05-636) and goat anti-mouse AF488-IgG (1:500; Jackson ImmunoResearch #115-545-062), followed by Hoechst 33342 for nuclei staining. Slides were imaged on an Eclipse Ti-E confocal microscope (Nikon) with a Plan Apo VC 60× oil DIC N2 objective (Nikon). Ten sections (0.4 μm thickness) were imaged. At least 30 nuclei/slide were imaged (2 slides/treatment). Maximal intensity projections of z-stacks were made using ImageJ v1.51p (http://imagej.nih.gov/ij). Nuclei and γH2AX foci were counted using CellProfiler v3.1.9 (http://cellprofiler.org) to determine the average numbers of γH2AX foci per nucleus in each image.