Gamma H2AX assay

To detect DNA double strand breaks (DSB), immunfluorescence staining of phosphorylated (Ser139) histone H2AX (gammaH2AX), an early DNA repair protein, was performed as described previously [24]. Therefore, GBM cells were seeded in 8-well chamber slides (2000–2500 cells/ well), allowed to adhere for 24 h, and irradiated (4 Gy) or THPTS-PDT treated, except for untreated controls. To allow for repair of potentially induced DSB, submaximal THPTS concentrations (DBTRG-05MG 1 μg/ml, U-87MG 2 μg/ml, A-172 5 μg/ml, 3 hours drug light interval) were used, inhibiting cell proliferation by 30% (IC 30). GammaH2AX staining was executed 1 or 16 hours after laser treatment. A donkey anti-mouse Cy5-labeled secondary antibody (4 μg/ml; Jackson ImmunoResearch) was used here contrary to Patties et al.