Optimized protocol for single-cell isolation and alkaline comet assay to detect DNA damage in cells of Drosophila wing imaginal discs

Reduced expression of nucleolar genes induces stress and DNA damage. Here, we present a protocol to analyze DNA fragmentation at the single-cell level in Drosophila imaginal discs using an optimized alkaline comet assay. We describe steps for larvae development, tissue disaggregation, and single-cell dissociation. We then detail procedures for cell lysis, electrophoresis, and DNA visualization. This approach provides insights into the molecular consequences of nucleolar stress induction and its role in the DNA damage response pathways in vivo.