TUNEL Labeling

TUNEL labeling may also show significant variation with different histological fixatives, mode of fixation, length of fixation time, and enzyme pretreatment, resulting in more than 10-fold different TUNEL positive rates [86, 94, 103-106],... 

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.)... 

The terminal dUTP nick end labeling assay (TUNEL reaction) and electrophoretic analysis of DNA/organotin(IV) mixtures allowed the investigation of DNA fragmentation. 

TUNEL Assay (Terminal Transferase dUTP Nick End Labelling)... 

TUNEL terminal transferase dUTP nick end labelling... 

Fig. 1. Labeling of degraded chromatin by the TUNEL assay. During apoptosis endogenous, endonucleases cleave chromatin in the hnker region between nucleosomes. The resulting nucleosome multimers are labeled by TdT and a dUTP analog with a detectable label (biotin, DIG, or FITC) shown as. The additional nucleotide in the reaction (here shown as dCTP) may be any dNTP and serves to extend the labeling reaction by preventing steric hindrance by two adjacent labeled dUTPs. (Abbreviations are as in text.)...

Fig. 2. Apoptotic cells fluorescently labeled using the TUNEL assay. Fixed paraffin-embedded rat mammary glands, 4 d postweaning (obtained from Oncor), were labeled using the TUNEL assay to detect apoptotic cells. Reagents were from Oncor s Apoptag Direct In Situ Apoptosis Detection Kit (Fluorescein), which directly incorporates fluorescein-labeled dUTP in the TdT end-labeling reaction. All reagents were used as described by the manufacturer (see Note 1). (Abbreviations are as in text.)...

Treatment of fixed cells with DNasel will provide positive controls for the TUNEL assay. DNasel will digest chromosomal DNA and provide 3 -OH ends for the TdT enzyme to label in the TUNEL reaction. Labeling of these control cells will appear diffuse throughout the nucleus. 

Additionally, two studies have measured colorectal epithelial cell proliferation and apoptosis in human non-neoplastic mucosa in combination with serum bile acid quantification. Ochsenkuhn et al have reported a positive correlation between serum DCA levels and proliferation measured by flow cytometric cell cycle analysis. However, a more recent study of colorectal adenoma patients failed to detect a correlation between serum DCA and immuno-histochemical Ki-67 antigen labelling. Instead, this latter study revealed a positive correlation between serum DCA and the degree of TUNEL-positive epithelial cell apoptosis. ... 

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

Fig. 7. Schematic representation of the principle of TUNEL assay. The enzyme TdT catalyzes a template-independent addition of bromolated deoxyuridine triphosphates (Br-dUTP) to the 3 -OH ends of double- and single-stranded DNA. After Br-dUTP incorporation, DNA break sites are identified by an FITC-labeled anti-BrdU monoclonal antibody.

Fig. 9. Flow analysis of apoptotic human peripheral blood lymphocytes using direct TUNEL assay. Human peripheral blood lymphocytes (1 x 10 ) treated (A) without or (B) with dexamethasone (0.1 ijlM) for 16 h were transferred to a 15-ml tube. Paraformaldehyde (2%) was added to cells with shaking and incubated for 10-15 min in ice with occasional shaking. Cells were washed with PBS with 1% BSA and 3-4 ml cold acetone was then added to cells. After 2-3 min incubation on ice with occasional shaking, cells were washed twice and TUNEL reaction mixture including enzyme TdT and fluorescein-labeled anti-dUTP antibody was added to cells (H). For the negative control group, only label solution without TdT was added to cells ( ). Cells without any addition of reaction or label solution were used for assessment of the autofluorescence ( ). The cell mixture was incubated 1 h at 37°C in the dark. The result of the apoptosis after flow analysis was expressed as a histogram using software CellQuest (Becton Dickinson). In Fig. 9A Ml (nonapoptotic cells), 86% M2 (apoptotic cells), 14%. In Fig. 9B Ml (nonapoptotic cells), 78% M2 (apoptotic cells), 22% (our unpublished data).

Fig. 8.24. Cells in late apoptosis have fragmented DNA. They can be visualized as cells with increased incorporation of fluorochrome-labeled nucleotides to the ends of the fragments in the flow cytometric TUNEL assay (lower dot plots, with FL1 as FITC-dUTP and FL2 as propidium iodide bound to DNA). Alternatively, they can be visualized as cells with less-than-normal (sub-GO/Gl) DNA content (upper histograms of propidium iodide fluorescence). Data from etoposide-treated ML-1 cells courtesy of Mary Kay Brown and Alan Eastman.

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. 

DNA damage was evaluated by the terminal deoxynucleotidyltransferase (TdT)-mediated UTP nick end labeling (TUNEL) assay. Degenerating cells were stained by the Fluoro-Jade dye. 

Cells positive for NeuN, TUNEL, and Fluoro-Jade were evaluated in grids of 800 pm x 500 pm (CAl) or 800 pm x 1,000 pm (neocortex and striatum). Areas were determined by public domain software (ImageJ http //rsb.info.nih.gov/ij). The number of positive cells was divided by the respective area (total area or frame area). Thus, single-labeled cells for any marker were quantified densitometrically (cells/mm2). Densities were averaged to obtain a mean density value for each region/animal group. 

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. 

Kanoh M, Takemura G, Misao J, Hayakawa Y, Aoyama T, Nishigaki K, et al. Significance of myocytes with positive DNA in situ nick end-labeling (TUNEL) in hearts with dilated cardiomyopathy Not apoptosis but DNA repair. Circulation 1999 99 2757-2764. 

CBF, Cerebral blood flow CCAo, common carotid artery occlusion cGMP, cyclic guanine monophosphate ICAM-1, intercellular adhesion molecule-1 ICAo, internal carotid artery occlusion MCAo, middle cerebral artery occlusion NAA, A-acetyl-aspartate NOS, nitric oxide synthase SD, Sprague-Dawley SH, spontaneously hypertensive SOD, superoxide dismutase TUNEL, transferase dUTP nick-end labeling. 

The mechanism of delayed neuronal injury following TBI includes apoptosis (63,64). Experimental data after cerebral hypoxic-ischemic injury indicate that moderate postinsult hypothermia (34°C) reduced the fraction of apoptotic cells but not cells undergoing necrosis (65). In another study, intraischemic hypothermia reduced the number of transferase dUTP nick-end labeling (TUNEL)-positive cells after transient focal ischemia (66). Thus, it will be important in future studies to determine whether hypothermia inhibits apoptotic neuronal cell death in models of TBI. 

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