Abstract
DNA- and histone-related research frequently comprises the quantitative analysis of protein modifications, such as histone phosphorylation. Analysis of accumulation and disappearance of protein foci are used to monitor DNA damage and repair kinetics. If the protein of interest doesn’t accumulate in foci, laser micro-irradiation of single nuclei provides an alternative method to monitor DNA repair proteins and histone dynamics at the DNA damage site. We have developed an automated evaluation tool for standardized, high-throughput analysis of micro-irradiated cells featuring single cell background subtraction and detection across multiple fluorescence channels, allowing for robust statistics.
Highlights
When characterizing the cellular DNA damage response to environmental compounds, chemotherapy, radiation, or other DNA damaging agents, nuclear protein localization is an effective and widely used method to analyse DNA repair kinetics by detecting changes in chromatin structure and protein localization
Laser micro-irradiation allows analysing the accumulation of the protein of interest or its modification, even if these events don’t manifest as foci and just result in an increased presence of the protein or protein modification at the DNA damage region[4,7,8,12]
We have developed an ImageJ-based, user-friendly, high-throughput evaluation tool, the Stripenator, to standardize and accelerate the quantitative analysis of local protein accumulation at sites of DNA damage
Summary
When characterizing the cellular DNA damage response to environmental compounds, chemotherapy, radiation, or other DNA damaging agents, nuclear protein localization is an effective and widely used method to analyse DNA repair kinetics by detecting changes in chromatin structure and protein localization. The analysis of large sets of images and the resulting datasets from cells with defined micro-irradiated areas is still challenging, time-consuming and often performed as qualitative analysis, not statistically meaningful, as there is no tool available for the analysis.
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