Apoptosis TUNEL

Maier et al., 1998 SD rats MCAo 2 h, with reperfusion 30, 33, and 37, during first 30 min, 1 h, or 2 h of ischemia Infarct size, neurologic function, apoptosis (TUNEL stain, morphology, DNA fragmentation), inflammation (MPO stain) 1 d and 3 d postreperfusion Reduced infarct size, improved neurologic function, reduced apoptosis and inflammation with 1 h or 2 h hypothermia... 

Royle et al. (2004) NO-NSAIDS Human prostate (LNCap and PC-3) Cell viability (MTT) and apoptosis (TUNEL) <80% growth inhibition by NO aspirin (NCX 4060) and <90% apoptotic cells ... 

Tessei et al. (2005) NO-NSAIDS Human colon (LoVo, LoVo Dx,WiDrandLRWZ) Cell growth (SRB assay) and apoptosis (TUNEL) Lowest IC50 10 p,M (NCX 4040) Human colon cancer cell lines (LoVo, LoVo Dx, WiDr) 40% reduction in tumour weight after NCX 4040 (10 mg/kg) 5 times/week for 6 weeks... 

Huerta et al. (2009) NONOate/cisplatin Human colon cancer (SW480 and SW620) Apoptosis (TUNEL) 1 mM DETANONOate increased >2-fold compared with cisplatin alone Human colon cancer (SW620) Growth delayed by 2 days compared with cisplatin alone No effect of DETANONOate alone (0.4mg/kg) every 2 days for 2 weeks)... 

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. 

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.)...

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. ... 

Furthermore, since the cell growth arrest is often linked to cell death. The annexin V staining positive cell or the amount of DNA fragmentation assessed by TUNEL and FACS analysis has been interpreted as indicative of apoptosis. The HDACI-induced apoptosis can also be determined by Western blotting of target proteins, detection of mitochondrial membrane potentials, activation of caspases and their substrate cleavages in a dose- and time-dependent manner. 

The combination of annexin V binding assay and the TUNEL method can reveal the presence of three subpopulations of apoptotic cells in tissues (1) annexin V-positive/TUNEL-negative cells which are in the early phase of apoptosis,... 

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.

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. 

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,... 

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. 

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