Fig. 5

Neutral comet assay detects no DSBs in primary cultures with elevated neuronal activity and pan-nuclear γH2AX elevation. a–c Cultures of primary hippocampal mouse neurons were assessed for DSB levels by the neutral comet assay (a, b) or immunocytochemistry (c). Cells were treated with vehicle (Ctrl), 10 μM bicuculline (Bic) or 5 μM etoposide (ETP) for 1 h before harvesting. a Representative images of cell nuclei stained with SYBR Green after electrophoresis at neutral pH. Insets show higher magnification views. Scale bar: 100 μm; 20 μm (insets). b Quantitation of comet tail lengths. A total of 150–200 nuclei were measured for each mouse and condition. n = 3 cultures/mice per condition. Circles indicate mean values for each mouse; grey dots indicate individual nuclei. ***p < 0.001 by one-way ANOVA and Holm-Sidak test. ns, not significant. c Representative confocal images of cultured neurons exposed to the indicated treatments. Cells were co-labeled for γH2AX (green), NeuN (red), and Hoechst 33342 (blue). Scale bar: 10 μm