title: Detection of DNA Fragmentation in Apoptotic Cells by TUNEL.
abstract: Degradation of DNA into oligonucleosomal-sized fragments is a unique event in apoptosis that is orchestrated by caspase-activated DNase. Traditionally, this event is observed by resolving cellular DNA by gel electrophoresis, which results in a characteristic "ladder" pattern. However, this technique is time-consuming and cannot be used to quantitate the number of apoptotic cells in a sample. Terminal dUTP nick-end labeling (TUNEL) of fragmented DNA allows researchers to identify DNA fragmentation at the single-cell level. This method involves the specific addition of fluorescently labeled UTP to the 3'-end of the DNA fragments by terminal deoxynucleotidyl transferase. The TUNEL assay is both fast and sensitive. Here, we describe a protocol in which cells are treated with TUNEL reagent and counterstained with Hoechst 33342. In contrast to TUNEL, which only stains apoptotic cells, Hoechst 33342 stains the DNA of all cells.
authors.affiliation: Apoptosis and Cytotoxicity Laboratory, Mater Research, Translational Research Institute, Woolloongabba, Brisbane, Queensland 4102, Australia.
authors.forename: Lisa C
authors.initials: LC
authors.lastname: Crowley
authors.affiliation: Apoptosis and Cytotoxicity Laboratory, Mater Research, Translational Research Institute, Woolloongabba, Brisbane, Queensland 4102, Australia.
authors.forename: Brooke J
authors.initials: BJ
authors.lastname: Marfell
authors.affiliation: Apoptosis and Cytotoxicity Laboratory, Mater Research, Translational Research Institute, Woolloongabba, Brisbane, Queensland 4102, Australia; Flow Cytometry and Imaging, QIMR Berghofer Medical Research Institute, Herston, Brisbane, Queensland 4006, Australia; School of Medicine, University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia.
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Waterhouse
mesh_terms: D017209:Apoptosis; D001562:Benzimidazoles / Q000378:metabolism; D003584:Cytological Techniques / Q000379:methods*; D053938:DNA Fragmentation; D005456:Fluorescent Dyes / Q000378:metabolism; D020287:In Situ Nick-End Labeling / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods; D013194:Staining and Labeling
chemical_list: D001562:Benzimidazoles; D005456:Fluorescent Dyes; C017807:bisbenzimide ethoxide trihydrochloride