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Updated: May 29, 2026
Cell Cycle-specific Measurement of γH2AX and Apoptosis After Genotoxic Stress by Flow Cytometry
Published on: September 1, 2019
Analyzing p53 regulated DNA damage checkpoints by flow cytometry
Lois Resnick-Silverman1, James J Manfredi
1Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY 10018, USA. lois.resnick-silverman@mssm.edu
Abstract:
The most critical feature of the cellular response to DNA damage is the ability of the cell to pause and repair the damage so that detrimental mutations will not be passed along to future generations of cells. The cell cycle of mammalian cells is equipped with checkpoints that can prevent cell cycle progression. Cells can either be arrested before replication of the DNA when the cells have a 2 N amount of DNA or after replication and prior to cell mitosis when the cells have a 4 N amount of DNA. Flow cytometry is a standard technique that is used to 'sort' cells based on their DNA content. It uses the principles of light scattering, light excitation, and emission of fluorochrome molecules to generate data about individual cells. The cells are fixed and permeabilized so that the DNA can be stained with a fluorescent dye. Cells that have a 2 N amount of DNA can be separated from cells with a 4 N amount of DNA. Using this technique, changes in the profile of the G1, S, and G2/M phases of the cell cycle are readily seen after DNA damage.
Insights
Cell cycle checkpoints pause cell division to repair DNA damage, preventing mutations. Flow cytometry analyzes DNA content to observe these cell cycle changes after damage.
Area of Science:
- Cell Biology
- Genetics
- Biotechnology
Background:
- Cellular response to DNA damage is critical for preventing mutations.
- Cell cycle checkpoints in mammalian cells halt progression to allow for DNA repair.
- Cells arrest at specific DNA content levels (2N or 4N) during the cell cycle.
Purpose of the Study:
- To explain the role of cell cycle checkpoints in DNA damage response.
- To describe how flow cytometry is utilized to analyze cell cycle progression.
- To demonstrate the application of flow cytometry in observing cell cycle alterations post-DNA damage.
Main Methods:
- Flow cytometry, a technique utilizing light scattering and fluorescence, is employed.
- Cells are fixed and permeabilized to enable DNA staining with fluorescent dyes.
- Separation of cells based on DNA content (2N vs. 4N) is achieved.
Main Results:
- Flow cytometry effectively sorts cells based on their DNA content.
- Changes in the distribution of G1, S, and G2/M phases are observable.
- The technique allows for the visualization of cell cycle arrest following DNA damage.
Conclusions:
- Flow cytometry is a valuable tool for studying the cell cycle response to DNA damage.
- Understanding cell cycle regulation is crucial for preventing genetic instability.
- This method facilitates the analysis of DNA repair mechanisms and their impact on cell division.
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