TUNEL assay

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) is a method for detecting DNA fragmentation by labeling the 3′- hydroxyl termini in the double-strand DNA breaks generated during apoptosis.^[1][2]

Mouse liver showing an apoptotic cell stained with TUNEL

Method

TUNEL is a method for detecting apoptotic DNA fragmentation, widely used to identify and quantify apoptotic cells, or to detect excessive DNA breakage in individual cells.^[3] The assay relies on the use of terminal deoxynucleotidyl transferase (TdT), an enzyme that catalyzes attachment of deoxynucleotides, tagged with a fluorochrome or another marker, to 3'-hydroxyl termini of DNA double strand breaks. It may also label cells having DNA damage by other means than in the course of apoptosis. ^{[citation needed]}

History

The fluorochrome-based TUNEL assay applicable for flow cytometry, combining the detection of DNA strand breaks with respect to the cell cycle-phase position, was originally developed by Gorczyca et al.^[4] Concurrently, the avidin-peroxidase labeling assay applicable for light absorption microscope was described by Gavrieli et al.^[5] Since 1992 the TUNEL has become one of the main methods for detecting apoptotic programmed cell death.^[6] However, for years there has been a debate about its accuracy, due to problems in the original assay which caused necrotic cells to be inappropriately labeled as apoptotic.^[7] The method has subsequently been improved dramatically and if performed correctly should only identify cells in the last phase of apoptosis.^[8][9] New methods incorporate the dUTPs modified by fluorophores or haptens, including biotin or bromine, which can be detected directly in the case of a fluorescently-modified nucleotide (i.e., fluorescein-dUTP), or indirectly with streptavidin or antibodies, if biotin-dUTP or BrdUTP are used, respectively. The most sensitive of them is the method utilizing incorporation of BrdUTP by TdT followed by immunocytochemical detection of BrdU.^[10]