FADU assay

Cells were seeded in 96-well round-bottom culture plates. The next day, cells were treated with 100 µM H₂O₂ for 30 min or 1 µM CPT for 60 min. Subsequently, treatment medium was exchanged for fresh cell culture medium and cells were either incubated for 60 or 240 min to allow time for DNA repair or directly used for further analysis. Therefore, cells were washed in 100 µL ice-cold PBS and the DNA integrity was determined via fluorometric detection of alkaline DNA unwinding (FADU). The following pipetting steps were automated by a liquid handling device. First, 7.5 µL PBS and 7.5 µL lysis buffer [400 mM guanidine thiocyanate, 30 mM EDTA, 0.2% N-lauroylsarcosyl pH 12], both supplemented with 300 µM DFA and 300 µM TEMPOL, were added to each well and plates were incubated for 12 min at 4 °C. After cell lysis, 270 µL dilution buffer [8.4 mM HEPES] was added, followed by incubation for 1 h at 37 °C. ‘T-value samples’ (T values reflect the amount of total DNA) were then treated with 150 µL neutralization buffer [1 M D-( +)-glucose monohydrate, 14 mM β-mercaptoethanol], before all samples were treated with 50 µL alkaline unwinding buffer [200 mM NaOH in 40% lysis buffer] and incubated for 1 h at 30 °C to allow for DNA unwinding. Following a cool-down to 22 °C for 30 min, ‘P-value samples’ (P-values serve as a reciprocal measure for cellular DNA strand breaks) were then also treated with 150 µL neutralization buffer. For fluorescence measurement, 50 µL SYBRGreen I solution (ThermoFisher Scientific; 1:8.333 in ddH₂O) were added to each well and the signal intensity was determined (λ_ex 485 nm, λ_em 535 nm) using an Infinite F200 plate reader (Tecan). The amount of double-stranded DNA compared to the whole DNA was calculated by dividing the mean of P-values by the mean of corresponding T-values (both measured in technical quadruplicates) (Mack et al. 2021; Moreno-Villanueva et al. 2009).