On the use of an appropriate TdT-mediated dUTP-biotin nick end labeling assay to identify apoptotic cells
| Autor: | Imene Jaadane, Francine Behar-Cohen, Cecile Lebon, Gloria E. Villalpando Rodriguez, Alicia Torriglia, Ikram El Zaoui |
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| Rok vydání: | 2015 |
| Předmět: |
Male
Programmed cell death Biophysics Biotin Apoptosis DNA Fragmentation Biology Biochemistry Mice In Situ Nick-End Labeling/methods Cricetinae In Situ Nick-End Labeling Animals Humans Phosphorylation Fragmentation (cell biology) Molecular Biology Cells Cultured Tissue homeostasis TUNEL assay Caspases/metabolism Apoptotic DNA fragmentation Cell Biology Molecular biology Rats Cell biology Mice Inbred C57BL Terminal deoxynucleotidyl transferase Rats Inbred Lew Caspases DNA fragmentation Deoxyuracil Nucleotides HeLa Cells |
| Zdroj: | Analytical Biochemistry, vol. 480, pp. 37-41 |
| Popis: | Apoptosis is an essential cellular mechanism involved in many processes such as embryogenesis, metamorphosis, and tissue homeostasis. DNA fragmentation is one of the key markers of this form of cell death. DNA fragmentation is executed by endogenous endonucleases such as caspase-activated DNase (CAD) in caspase-dependent apoptosis. The TUNEL (TdT-mediated dUTP-biotin nick end labeling) technique is the most widely used method to identify apoptotic cells in a tissue or culture and to assess drug toxicity. It is based on the detection of 3'-OH termini that are labeled with dUTP by the terminal deoxynucleotidyl transferase. Although the test is very reliable and sensitive in caspase-dependent apoptosis, it is completely useless when cell death is mediated by pathways involving DNA degradation that generates 3'-P ends as in the LEI/L-DNase II pathway. Here, we propose a modification in the TUNEL protocol consisting of a dephosphorylation step prior to the TUNEL labeling. This allows the detection of both types of DNA breaks induced during apoptosis caspase-dependent and independent pathways, avoiding underestimating the cell death induced by the treatment of interest. |
| Databáze: | OpenAIRE |
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