TUNEL assay

CLARKE R G, LUND E K, JOHNSON I T aud PINDER A c (2000) Apoptosis cau he detected in attached colonic adenocarcinoma HT29 cells using annexin V binding, hut uot hy TUNEL assay or suh-GO DNA content . Cytometry, 39 141-50. 

Apoptosis or programmed cell death is one of the regulatory mechanisms for the removal of unwanted cells. Apoptosis is induced by the stimulation of several different cell surface receptors in association with caspase activation. Apoptosis of a cell is thus a complicated process and can be assayed by various methods. Among widely used methods, the TUNEL assay is described here. 

TUNEL Assay (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.)...

Proceed with TUNEL Assay for Adherent Cells (Subheading 3.5.2.). 

Proceed with TUNEL assay for Cell Suspensions for Flow Cytometry (Subheading 3.5.1.)... 

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. 

Proceed with the TUNEL Assay for Adherent Cells (Subheading 3.5.2.). Process all positive control slides in separate coplin jars to avoid introducing DNasel contamination into experimental samples. 

Moved] Cranberry fruit of Early Black cultivar was fractionated chromatographically and fractions were analyzed for flavonoid content. The effects of the flavonoid fractions and ursolic acid, an abundant triterpenoid in cranberry peel, were assessed in two models of colon cancer and one model of breast cancer. Clonogenic soft agar assays were used to determine the effect of these compounds on tumor colony formation in HCT-116, HT-29 and MCF-7 cells. MTT and trypan blue assays were performed to assess their ability to inhibit tumor cell proliferation. TUNEL assays were performed to assess apop-totic response to the cranberry compounds. The proanthocyanidins inhibited tumor colony formation in HCT-116 and HT-29 cells in a dose-dependent manner, with greater effect on the HCT-116 cell line. Ursolic acid strongly inhibited tumor colony formation in both colon cell lines. These compounds also decreased proliferation in all three tumor cell lines with the HCT-116 cell line most strongly affected. (150 words)... 

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. 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.

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).

DNA fragmentation 4.8 Apoptotic DNA ladder assay TUNEL assay... 

Tumors, 110, 119, 121, 121f, 127f, 128, 130, 145, 186-189, 187f see also Malignancy/cancer TUNEL assay, 153-154, 154f, 256... 

LIGHT SCATTER RHODAMINE 123, DiOC(6)3, or JC-1 with ANNEXIN V and PI with TUNEL ASSAY eg SNARF... 

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. 

Fig. 3A-D Postischemic damage in CA1 on day 4. A Staining for the TUNEL assay showing numerous positive cells. B Statistical analysis of TUNEL+ cells per frame of 0.4 mm2 in CA1 p < 0.001, one-way ANOVA followed by Tukey-Kramer post hoc. C Staining for Fluoro-Jade demonstrating similar pattern to the TUNEL stain. D Statistical analysis of Fluoro-Jade+ cells per frame of 0.4 mm2 in CA1 p < 0.001, one-way ANOVA followed by Tukey-Kramer post hoc. Scale bar = 200 pm...

Second, BrdU was not detected in cells exhibiting features of DNA damage, repair, or programmed cell death. DNA damage was detected using the TUNEL assay. As expected, ischemia increased the number of TUNEL+ cells in several... 

After behavioral evaluation in the 8-arm radial maze task, the rat s brain was fixed and apoptosis was identified in the hippocampal CA1 by TUNEL assay. Apoptotic cells were found in vehicle-treated rats on the 7th day after ischemia (mean F S.E.M. 78.4 F 5.7 TUNEL-positive cells/mm2). As shown in Fig. 8,... 

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 1465-1468. 

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