TUNEL assay

[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.

[citation needed] 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.