Chromatid Break and SCE Analysis

For PCC analysis, cells were treated with 50 ng/ml calyculin A (Calbiochem) for 30 min before harvesting. For SCE analysis, cells were grown for 48 h in BrdU before irradiation. Colcemid (together with 1 mM caffeine to overcome the G2/M arrest) was added at 8 h until 12 h post-irradiation to collect cells in mitosis. Chromatid breaks (for PCC analysis) or SCEs were scored in at least 100 chromosome spreads from at least three independent experiments per data point. Staining was according to standard protocols. Aphidicolin (Sigma) was added at 1 μg/ml immediately before IR. ATM inhibitor (KU55933) and DNA-PK inhibitor (NU7026), a kind gift from Kudos Pharmaceuticals (Cambridge, UK), were added at 20 μM 60 min prior to IR. X-ray irradiation was performed at 90- or 120-kV γ-irradiation using a ¹³⁷Cs source. Dosimetry was performed with ion chambers and considered the increase in dose for cells grown on glass coverslips relative to plastic surfaces (Kegel et al, 2007 (link)).