title: On the use of an appropriate TdT-mediated dUTP-biotin nick end labeling assay to identify apoptotic cells.
abstract: 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.
authors: Centre de Recherches des Cordeliers, INSERM U1138, Université Pierre et Marie Curie, Université Paris Descartes, 75006 Paris, France.
authors: Cecile
authors: C
authors: Lebon
authors: Centre de Recherches des Cordeliers, INSERM U1138, Université Pierre et Marie Curie, Université Paris Descartes, 75006 Paris, France.
authors: Gloria Villalpando
authors: GV
authors: Rodriguez
authors: Centre de Recherches des Cordeliers, INSERM U1138, Université Pierre et Marie Curie, Université Paris Descartes, 75006 Paris, France.
authors: Ikram El
authors: IE
authors: Zaoui
authors: Centre de Recherches des Cordeliers, INSERM U1138, Université Pierre et Marie Curie, Université Paris Descartes, 75006 Paris, France.
authors: Imene
authors: I
authors: Jaadane
authors: Centre de Recherches des Cordeliers, INSERM U1138, Université Pierre et Marie Curie, Université Paris Descartes, 75006 Paris, France.
authors: Francine
authors: F
authors: Behar-Cohen
authors: Centre de Recherches des Cordeliers, INSERM U1138, Université Pierre et Marie Curie, Université Paris Descartes, 75006 Paris, France. Electronic address: alicia.torriglia@inserm.fr.
authors: Alicia
authors: A
authors: Torriglia
mesh_terms: D000818:Animals; D017209:Apoptosis; D001710:Biotin; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D053938:DNA Fragmentation; D003856:Deoxyuracil Nucleotides; D006367:HeLa Cells; D006801:Humans; D020287:In Situ Nick-End Labeling / Q000379:methods*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010766:Phosphorylation; D051381:Rats; D011917:Rats, Inbred Lew
chemical_list: D003856:Deoxyuracil Nucleotides; C027078:deoxyuridine triphosphate; D001710:Biotin; D020169:Caspases
keywords: Apoptosis; Caspase-independent cell death; Endonuclease; LEI/L-DNase II; TUNEL assay