Related Experiment Videos
A cautionary note on the use of the TUNEL stain to determine apoptosis
C Charriaut-Marlangue1, Y Ben-Ari
1Université R. Descartes, INSERM U 29, Paris, France.
Abstract:
Detection of DNA fragments in situ using the terminal deoxyribonucleotidyl transferse (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL assay) is now commonly used to investigate apoptosis. Previous reports suggested that physiologically appropriate death is due to apoptosis and that pathological mechanisms involve necrosis. Strong evidence of apoptosis, following ischaemia and epilepsy, has been recently provided by combining genomic DNA gel electrophoresis, light and electron microscopy and in situ, DNA-break labelling. However, only an observation in light microscopy with high magnification permits the detection of chromatin condensation and apoptotic bodies. We report that a positive TUNEL assay reaction should not be considered as a specific marker of apoptosis but can also indicate necrotic cell death.
Insights
The TUNEL assay detects DNA breaks, commonly used for apoptosis. However, this study shows a positive TUNEL reaction can also indicate necrotic cell death, not just apoptosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Pathology
Background:
- The terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL) assay is widely used to detect DNA fragmentation in situ, a hallmark of apoptosis.
- While physiological cell death is often attributed to apoptosis and pathological cell death to necrosis, distinguishing between these processes can be challenging.
- Previous research has provided evidence for apoptosis in conditions like ischemia and epilepsy using various techniques, including DNA gel electrophoresis, microscopy, and in situ DNA-break labeling.
Purpose of the Study:
- To evaluate the specificity of the TUNEL assay in distinguishing between apoptosis and necrosis.
- To investigate whether a positive TUNEL assay reaction is exclusively indicative of apoptosis.
Main Methods:
- Utilized the terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL) assay for in situ detection of DNA fragmentation.
- Correlated TUNEL assay results with light microscopy observations, specifically focusing on chromatin condensation and apoptotic bodies.
- Reviewed existing literature on apoptosis and necrosis detection methods.
Main Results:
- A positive TUNEL assay reaction indicates the presence of DNA breaks.
- However, the study found that a positive TUNEL assay is not a specific marker solely for apoptosis.
- Necrotic cell death can also result in DNA fragmentation detectable by the TUNEL assay.
Conclusions:
- The TUNEL assay detects DNA strand breaks, which occur in both apoptosis and necrosis.
- Therefore, a positive TUNEL assay result should not be exclusively interpreted as evidence of apoptosis.
- Further investigation, including high-magnification light microscopy, is necessary to differentiate between apoptotic and necrotic cell death when using the TUNEL assay.