Protocol for single-cell analysis of DNA double-strand break production and repair in cell-cycle phases by automated high-content microscopy
Year: 2025
Type: article
Abstract: The mechanisms of DNA double-strand break (DSB) production and repair vary throughout the cell cycle. Here, we provide a protocol to quantify DSB production and repair in G1, S, and G2 phases of asynchronous adherent cells by coupling the staining of DSBs and cell-cycle markers with automated high-content fluorescence microscopy. We describe steps for cell seeding, treatment, staining, imaging, and analysis. This protocol is broadly applicable for monitoring DSB dynamics at single-cell level throughout the cell cycle. For complete details on the use and execution of this protocol, please refer to Geraud et al. 1 • Steps for quantification of DSB production and repair in cell-cycle phases • Instructions for automated high-content imaging • Guidance on cell seeding, staining, and data analysis Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The mechanisms of DNA double-strand break (DSB) production and repair vary throughout the cell cycle. Here, we provide a protocol to quantify DSB production and repair in G1, S, and G2 phases of asynchronous adherent cells by coupling the staining of DSBs and cell-cycle markers with automated high-content fluorescence microscopy. We describe steps for cell seeding, treatment, staining, imaging, and analysis. This protocol is broadly applicable for monitoring DSB dynamics at single-cell level throughout the cell cycle.
Source: STAR Protocols
Language: English
Topic: DNA Repair Mechanisms
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,550
Awards: G133319545, G3997468695, Cassures double-brin de l'ADN transcriptionnelles par les ligands de G4s : mécanismes moléculaires de production et de réparation et pertinence pour les cancers déficients en BRCA1/2, G5836999278, G6098272583, Transcription-associated genomic instability in the acquisition of secondary resistances to targeted therapies in lung cancer, G6546605005, G6712696899, G7222423151, G8598742569
OpenAlex ID: W4408044353