Terminal deoxynucleotidyl transferase dUTP nick

There are several existing techniques to measure apoptosis in vitro, such as caspase activity assays, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), DNA fragmentation,... 

Tunel (Terminal deoxynucleotide transferase dUTP nick end labeUng) assay In a modified version of the Tunel assay, apoptotic ceUs can be measured in a two-color system using flow cytometry and microscopy. In this assay, DNA breaks are labeled by deoxynucleotidyl transferase using bromodeoxyuridine triphosphate (BrdUTP) and visualized by anti-BrdU antibody. Propidium iodide is used to counterstain the total ceUular DNA. 

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling... 

Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay examining apoptosis in smooth muscle cells and HL60 leukemia cells treated with paclitaxel. Abbreviations HL60, human leukemia 60. PTX, paclitaxel SMC, smooth muscle cells Tunel, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling. Source From Ref. 49. 

TUNEL stands for terminal deoxynucleotidyl transferase x-dUTP nick end labeling. This assay is based on the detection of DNA fragments marked by an enzyme that incorporates modified nucleotides to the 3 -OH ends of the fragments, which can be then specifically detected. The enzyme is a deoxynucleotidyl transferase, which can act in absence of a complementary strand. Among the nucleotides, there is one specifically marked with a fluorochrome, an enzyme, or an antigen. This allows different methods of detection. 

Biochemically, apoptosis is characterized by the internucleosomal degradation of chromosomal DNA to form a series of double-stranded fragments that are multiples of 180 200 base pairs in length. These fragments give a characteristic DNA ladder pattern on gel electrophoresis [91, 92] and can be detected by several cytochemical methods, the most extensively used being the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated dUTP nick end labeling (TUNEL) [93-95], The detection of ladder pattern and TUNEL positivity has been adopted as a marker of apoptosis. 

TUNEL terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick end-labeling assay... 

Fig. 2. Rat mammary gland, 3-4 d postweaning, stained by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. The nuclei of the apoptotic cells are stained dark. Nonapoptotic cell nuclei are counters tained with hematoxylin (pale).

FIGURE 8.5 Proapoprotic experimental therapies in pulmonary arterial hypertension (PAH). A common denominator of these therapies is the induction of apoptosis as well as suppression of proliferation in the pulmonary artery wall. On the left are untreated rats with monocrotaline-induced PAH and on the right are rats treated with elastase inhibitors. Increased apoptosis of the intima-media is showed by TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling), and decreased proliferation is imaged by staining for proliferation cell nuclear antigen (PCNA). The same pattern has been shown in the media of treated rats by all the therapies shown in the box. (From Ref. 116, with permission.)... 

Seven-week-old Wistar rats at normal iodine basehne were divided into four different groups and fed on water of normal, 1.5-fold, 3-fold and 6-fold iodine levels for 8 months. Transmission electron microscope, flow cytometry, DNA quantitation and annexin V early apoptosis detection technique were combined to evaluate intracellular ROS level expression of apoptosis-related molecules (Fas, FasL, bcl-2, bax) was traced by indirect fluorescent stained flow cytometry and immunohistochemistry qualitative and quantitative analyses of cell apoptosis were performed by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining. 

It has been shown that CLA was able to protect against 2-amino-3-methylimida-zo[4,5-/]quinoline-induced colon carcinogenesis in F344 rats (25). A recent study (26) demonstrated that 1% dietary CLA significantly decreased colon tumor incidence in the colon of 1,2-dimethylhydrazine (DMH)-treated rats, and the apoptotic index estimated by the terminal deoxynucleotidyl transferase-mediated dUTP nick... 

Another common method for measuring apoptosis is the DNA labeling technique, terminal deoxytransferase-mediated dUTP nick-end labeling (TUNEL). In this method, labeling of DNA strand breaks at the 3 end is achieved by using the enzyme, terminal deoxynucleotidyl transferase (TdT) and either a radioactive or a fluorescently-Iabeled deoxynucleotide. This method provides a very sensitive measurement of DNA strands breaks. However, as DNA strand... 

Although more labor intensive than the acridine orange method, the TUNEL-labeling technique (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labeling) provides a permanent record for labeling dead cells. (From Gavrieli et al. 1992 White et al. 1996 protocol and additional modifications were provided by P. Kurada and Kristen White.)... 

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