Rapid Detection of γ-H2Av Foci in Ex Vivo MMS-Treated Drosophila Imaginal Discs

Abstract

In Drosophila melanogaster, DNA double-strand breaks (DSBs) created by exposure to gamma or X-ray radiation can be quantified by immunofluorescent detection of phosphorylated histone H2Av (γ-H2Av) foci in imaginal disc tissues. This technique has been less useful for studying DSBs in imaginal discs exposed to DSB-inducing chemicals, since standard protocols require raising larvae in food treated with liquid chemical suspensions. These protocols typically take 3–4 days to complete and result in heterogeneous responses that do not provide information about the kinetics of DSB formation and repair. Here, we describe a novel and rapid method to quantify DSBs in imaginal discs cultured ex vivo with methyl methanesulfonate (MMS) or other DSB-inducing chemicals. The described method requires less than 24 h and provides precise control over MMS concentration and exposure time, enabling reproducible detection of transient DSBs. Furthermore, this technique can be used for nearly any chemical treatment and can be modified and adapted for several different experimental setups and downstream molecular analyses.