Quantifying Telomeric DNA Damage

Flow-sorted CD56^dim and CD56^neg NK cells were cytocentrifuged onto poly-L-lysine coated glass slides (Cytospin, Thermo Scientific). Staining for telomere-associated γH2A.X foci (TAF) was then performed as previously described [42 (link)]. Slides were air dried prior to hybridisation with 40 pM PNA probe targeting the TelC telomeric repeat (Panagene, TelC Cy3, #14 1224PL-01) for 2 hours. Sections were then counter stained for γH2A.X (Ser139, Cell Signaling #9718), followed by incubation with biotinylated secondary antibody (BA-1000, Vector) and FITC-streptavidin (A-2011). Slides were subsequently washed in formamide/SSC buffer prior to mounting with Vectorshield/DAPI (Vector Laboratories). Imaging was performed using a Leica SPE2 confocal microscope (Leica Microsystem). Analysis was performed using Fiji image analysis software (Fiji.sc).