TUNEL assay explained

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]}

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.

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]

References

1. Gorczyca W, Traganos F, Jesionowska H, Darzynkiewicz Z . 1993 . Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells. Analogy to apoptosis of somatic cells . Exp Cell Res . 207 . 1. 202–205 . 10.1006/excr.1993.1182 . 8391465 .
2. Lozano GM, Bejarano I, Espino J, González D, Ortiz A, García JF, Rodríguez AB, Pariente JA . 2009 . Density gradient capacitation is the most suitable method to improve fertilization and to reduce DNA fragmentation positive spermatozoa of infertile men . Anatolian Journal of Obstetrics & Gynecology . 3 . 1 . 1–7.
3. Lozano GM, Bejarano I, Espino J, González D, Ortiz A, García JF, Rodríguez AB, Pariente JA . 2009 . Relationship between Caspase Activity and Apoptotic Markers in Human Sperm in Response to Hydrogen Peroxide and Progesterone . Journal of Reproduction and Development . 55 . 6. 615–621 . 19734695 . 10.1262/jrd.20250 . free .
4. Gorczyca W, Bruno S, Darzynkiewicz RJ, Gong J, Darzynkiewicz Z . 1992 . DNA strand breaks occurring during apoptosis: Their early in situ detection by the terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors. . International Journal of Oncology. 1 . 6. 639–648 . 10.3892/ijo.1.6.639 . 21584593 .
5. Gavrieli Y, Sherman Y, Ben-Sasson SA. 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation . J. Cell Biol. . 1400587 . 2289665 . 10.1083/jcb.119.3.493 . 119 . 3. 493–501.
6. Darzynkiewicz Z, Galkowski D, Zhao H . 2008 . Analysis of apoptosis by cytometry using TUNEL assay . Methods . 44 . 3. 250–254 . 10.1016/j.ymeth.2007.11.008 . 18314056 . 2295206 .
7. Grasl-Kraupp B, Ruttkay-Nedecky B, Koudelka H, Bukowska K, Bursch W, Schulte-Hermann R . In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note . Hepatology . 1995 . 21 . 5 . 1465–8 . 7737654 . 10.1002/hep.1840210534. 28119339 . free .
8. Negoescu A, Lorimier P, Labat-Moleur F, Drouet C, Robert C, Guillermet C, Brambilla C, Brambilla E . In situ apoptotic cell labeling by the TUNEL method: improvement and evaluation on cell preparations . J Histochem Cytochem . 1996 . 44 . 9 . 959–68 . 8773561 . 10.1177/44.9.8773561. free .
9. Negoescu A, Guillermet C, Lorimier P, Brambilla E, Labat-Moleur F . Importance of DNA fragmentation in apoptosis with regard to TUNEL specificity . Biomed Pharmacother . 1998 . 52 . 6 . 252–8 . 9755824 . 10.1016/S0753-3322(98)80010-3.
10. Li X, Darzynkiewicz Z . 1995 . Labeling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation . Cell Proliferation . 28 . 11. 571–579 . 10.1111/j.1365-2184.1995.tb00045.x . 8555370 . 28993497 .