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Updated: Nov 4, 2025
Detection of Nuclear Blebbing and DNA Leakage in Mammalian Cells by Immunofluorescence
Published on: January 17, 2025
Immunofluorescence microscopy-based assessment of cytosolic DNA accumulation in mammalian cells
Ai Sato1, Aitziber Buque1, Takahiro Yamazaki1
1Department of Radiation Oncology, Weill Cornell Medical College, New York, NY 10065, USA.
Abstract:
Here, we describe an immunofluorescence (IF) microscopy-based approach to quantify cytosolic double-stranded DNA molecules in cultured eukaryotic cells upon the selective and specific permeabilization of plasma membranes. This technique is compatible with widefield microscopy coupled with automated image analysis for mid- to high-throughput applications and high-resolution confocal microscopy for subcellular assessments and co-localization studies. In addition to enabling single-cell and subcellular resolution, this approach circumvents most constraints associated with alternative approaches based on subcellular fractionation. For complete use and execution of this protocol, please refer to Yamazaki et al. (2020).
Insights
This study introduces a new immunofluorescence microscopy method to measure cytosolic double-stranded DNA in cells. The technique offers high resolution and throughput, overcoming limitations of other methods.
Area of Science:
- Cell Biology
- Molecular Biology
- Microscopy Techniques
Background:
- Cytosolic double-stranded DNA (dsDNA) is a key indicator of cellular stress and pathogen presence.
- Quantifying cytosolic dsDNA is crucial for understanding innate immune responses.
- Existing methods for dsDNA quantification have limitations in resolution and throughput.
Purpose of the Study:
- To develop and validate a novel immunofluorescence (IF) microscopy-based approach.
- To enable accurate quantification of cytosolic dsDNA in cultured eukaryotic cells.
- To provide a high-resolution, high-throughput alternative to existing methods.
Main Methods:
- Selective permeabilization of plasma membranes to access cytosolic dsDNA.
- Immunofluorescence (IF) microscopy using specific antibodies against dsDNA.
- Automated image analysis for widefield microscopy (mid- to high-throughput).
- High-resolution confocal microscopy for subcellular localization studies.
Main Results:
- The IF microscopy approach successfully quantifies cytosolic dsDNA.
- The technique is compatible with both widefield and confocal microscopy.
- It allows for single-cell and subcellular resolution.
- The method circumvents constraints of subcellular fractionation techniques.
Conclusions:
- This immunofluorescence microscopy protocol provides a robust and versatile method for cytosolic dsDNA quantification.
- It is suitable for various applications, from high-throughput screening to detailed subcellular analysis.
- The approach offers significant advantages over traditional methods, enhancing the study of DNA sensing pathways.
