Terminal deoxynucleotidyl transferase TUNEL

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) Assay... 

Fig. 8. Morphological changes of apoptotic eosinophils induced by dexamethasone (Z2). After eosinophils were treated (a) without or (b) with dexamethasone (2 /u,M) for 12 h, cells were harvested and detected by TUNEL assay using the In Situ Cell Death Detection Kit (Boehringer Mannheim). Briefly, cells were fixed with 4% paraformaldehyde and permeabilized by proteinase K and incubated with the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT). After washing to remove unbound enzyme conjugated antibody, the horseradish peroxidase retained in the immune complex was visualized by a substrate reaction with diaminobenzidine. The cell nucleus was counterstained with methanol green. Apoptotic eosinophils with nuclear DNA breaks were seen to stain dark brown using a Nikon Eclipse E800 microscope (Nikon Corporation, Tokyo, Japan) in Fig. 8b.

TUNEL The TUNEL assay represents a historical low point in attempts to coin acronyms. The Terminal deoxynucleotidyl transferase mediated dC/TP Nick End-Labeling assay labels the ends of DNA with fluorescent UTP. Apoptotic cells often have fragmented DNA these fragments will provide more substrate for the enzyme terminal deoxynucleotidyl transferase. Apoptotic cells can therefore be enumerated by flow cytometry according to their increased intensity in the TUNEL assay. 

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. 

Figure 29.8. Kidney tissue from a rat treated with para-aminophenol (300mg/kgip). Tissue was stained with hematoxylin and eosin and was subjected to terminal deoxynucleotidyl transferase-diaminobenzidine (TUNEL) staining to reveal DNA strand breaks. Darker gray areas represent tubules that stain positive for DNA strand breaks. Final magnification was lOOx (left panel) and 360x (right panel).

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

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,... 

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. 

A very sensitive method is the TUNEL assay (terminal deoxynucleotidyl transferase mediated dUTP-biotin end-labelling), that allows the direct... 

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... 

DNA fragmentation Biotin conjugated dUTP using TUNEL reaction terminal deoxynucleotidyl-transferase dUTP Optometric density... 

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.)... 

Sections (from Pll and P17 rats) are dewaxed in xylene, rehydrated in a decreasing ethanol series, rinsed in PBS, and incubated with the permeabilization solution for 8 min at room temperature. After two rinses in PBS (5 min each), slides are incubated with 50 pL of TUNEL mixture conjugated with FITC (5 pL terminal deoxynucleotidyl transferase solution and 45 pL label solution) according to the manufacturer s instructions for 3 h at 37 °C. 

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. 

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