Detection of Apoptosis by TUNEL Assay

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay is a long-established assay used to detect cell death-associated DNA fragmentation (3’-OH DNA termini) by endonucleases. Because these enzymes are particularly active in the kidney, TUNEL is widely used to identify and quantify DNA fragmentation and cell death in cultured kidney cells and animal and human kidneys resulting from toxic or hypoxic injury. The early characterization of TUNEL as an apoptotic assay has led to numerous misinterpretations of the mechanisms of kidney cell injury. Nevertheless, TUNEL is becoming increasingly popular for kidney injury assessment because it can be used universally in cultured and tissue cells and for all mechanisms of cell death. Furthermore, it is sensitive, accurate, quantitative, easily linked to particular cells or tissue compartments, and can be combined with immunohistochemistry to allow reliable identification of cell types or likely mechanisms of cell death. Traditionally, TUNEL analysis has been limited to the presence or absence of a TUNEL signal. However, additional information on the mechanism of cell death can be obtained from the analysis of TUNEL patterns.

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The developing ovary of the South American plains vizcacha, Lagostomus maximus (Mammalia, Rodentia): massive proliferation with no sign of apoptosis-mediated germ cell attrition

Reproduction ◽

10.1530/rep-10-0463 ◽

2011 ◽

Vol 141 (5) ◽

pp. 633-641 ◽

Cited By ~ 26

Author(s):

N P Leopardo ◽

F Jensen ◽

M A Willis ◽

M B Espinosa ◽

A D Vitullo

Keyword(s):

Germ Cell ◽

Germ Cells ◽

Ovarian Tissue ◽

Tunel Assay ◽

South American ◽

Follicular Atresia ◽

The South ◽

Cell Degeneration ◽

Lagostomus Maximus ◽

Apoptotic Activity

Apoptosis-dependent massive germ cell death is considered a constitutive trait of the developing mammalian ovary that eliminates 65–85% of the germinal tissue depending on the species. After birth and during adult lifetime, apoptotic activity moves from the germ cell proper to the somatic compartment, decimating germ cells through follicular atresia until the oocyte reserve is exhausted. In contrast, the South American rodent Lagostomus maximus shows suppressed apoptosis-dependent follicular atresia in the adult ovary, with continuous folliculogenesis and massive polyovulation, which finally exhausts the oocyte pool. The absence of follicular atresia in adult L. maximus might arise from a failure to move apoptosis from the germinal stratum to the somatic compartment after birth or being a constitutive trait of the ovarian tissue with no massive germ cell degeneration in the developing ovary. We tested these possibilities by analysing oogenesis, expression of germ cell-specific VASA protein, apoptotic proteins BCL2 and BAX, and DNA fragmentation by TUNEL assay in the developing ovary of L. maximus. Immunolabelling for VASA revealed a massive and widespread colonisation of the ovary and proliferation of germ cells organised in nests that disappeared at late development when folliculogenesis began. No sign of germ cell attrition was found at any time point. BCL2 remained positive throughout oogenesis, whereas BAX was slightly detected in early development. TUNEL assay was conspicuously negative throughout the development. These results advocate for an unrestricted proliferation of germ cells, without apoptosis-driven elimination, as a constitutive trait of L. maximus ovary as opposed to what is normally found in the developing mammalian ovary.

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TUNEL Assay for Identifying Ablated Myocardium

Pacing and Clinical Electrophysiology ◽

10.1111/j.1540-8159.2010.02883.x ◽

2010 ◽

Vol 33 (12) ◽

pp. 1548-1549 ◽

Cited By ~ 1

Author(s):

BRION WINSTON ◽

MICHAEL NALESNIK ◽

RAVEEN BAZAZ

Keyword(s):

Tunel Assay

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Cytotoxic Effect of Commercially Available Methylprednisolone Acetate with and without Reduced Preservatives on Dorsal Root Ganglion Sensory Neurons in Rats

January 2018 - Pain Physician ◽

10.36076/ppj.2014/17/e609 ◽

2014 ◽

Vol 5;17 (5;9) ◽

pp. E609-E618

Author(s):

Nebojsa N. Knezevic

Keyword(s):

Polyethylene Glycol ◽

Dorsal Root Ganglion ◽

Sensory Neurons ◽

Dorsal Root ◽

Cytotoxic Effect ◽

Caspase 3 ◽

Tunel Assay ◽

Methylprednisolone Acetate ◽

Drg Neurons ◽

Isolated Rat

Background: Epidural and intrathecal injections of methylprednisolone acetate (MPA) have become the most commonly performed interventional procedures in the United States and worldwide in the last 2 decades. However neuraxial MPA injection has been dogged by controversy regarding the presence of different additives used in commercially prepared glucocorticoids. We previously showed that MPA could be rendered 85% free of polyethylene glycol (PEG) by a simple physical separation of elements in the suspension. Objective: The objective of the present study was to explore a possible cytotoxic effect of commercially available MPA (with intact or reduced preservatives) on rat sensory neurons. Methods: We exposed primary dissociated rat dorsal root ganglia (DRG) sensory neurons to commercially available MPA for 24 hours with either the standard (commercial) concentration of preservatives or to different fractions following separation (MPA suspension whose preservative concentration had been reduced, or fractions containing higher concentrations of preservatives). Cells were stained with the TUNEL assay kit to detect apoptotic cells and images were taken on the Bio-Rad Laser Sharp-2000 system. We also detected expression of caspase-3, as an indicator of apoptosis in cell lysates. Results: We exposed sensory neurons from rat DRG to different concentrations of MPA from the original commercially prepared vial. TUNEL assay showed dose-related responses and increased percentages of apoptotic cells with increasing concentrations of MPA. Increased concentrations of MPA caused 1.5 – 2 times higher caspase-3 expression in DRG sensory neurons than in control cells (ANOVA, P = 0.001). Our results showed that MPA with reduced preservatives caused significantly less apoptosis observed with TUNEL assay labeling (P < 0.001) and caspase-3 immunoblotting (P ≤ 0.001) than in neurons exposed to MPA from a commercially prepared vial or “clear phase” that contained higher concentrations of preservatives. Even though MPA with reduced preservatives caused 12.5% more apoptosis in DRG sensory neurons than in control cells, post hoc analysis showed no differences between these 2 groups. Limitations: Our data was collected from in vitro isolated rat DRG neurons. There is a possibility that in vivo neurons have different extents of vulnerability compared to isolated neurons. Conclusions: Results of the present study identified a cytotoxic effect of commercially available MPA with preservatives or with a “clear phase” containing higher concentrations of preservatives on primary isolated rat DRG sensory neurons. This was shown by TUNEL positive assay and by increased caspase-3 expression as one of the final executing steps in apoptotic pathways in DRG neurons. However, our results showed no statistically significant difference between the control cells (salinetreated) and cells treated with MPA with reduced concentrations of preservatives, pointing out that either PEG or myristylgamma-picolinium chloride (MGPC) or their combination have harmful effects on these cells. Reduction of concentrations of preservatives from commercially available MPA suspensions by using the simple method of inverting vials for 2 hours could be considered useful in clinical practice to enhance the safety of this depot steroid when injected neuraxially. Key words: Methylprednisolone acetate, preservatives, dorsal root ganglion sensory neurons, cytotoxic effect, polyethylene glycol, myristylgamma-picolinium chloride

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Protective Effect of Penetratin Analogue-Tagged SOD1 on Cisplatin-Induced Nephrotoxicity through Inhibiting Oxidative Stress and JNK/p38 MAPK Signaling Pathway

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/5526053 ◽

2021 ◽

Vol 2021 ◽

pp. 1-13

Author(s):

Xiao-lu Wang ◽

Liang Wang ◽

Fo-lan Lin ◽

Si-si Li ◽

Ting-xuan Lin ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Membrane ◽

Signaling Pathway ◽

P38 Mapk ◽

Cell Apoptosis ◽

Mapk Signaling ◽

Tunel Assay ◽

Mapk Signaling Pathway ◽

Mapk Activation ◽

Urea Nitrogen

Copper/zinc superoxide dismutase (SOD1) can clear cisplatin- (CP-) induced excessive reactive oxygen species (ROS), but exogenous SOD1 cannot enter cells because of its low biomembrane permeability. Cell-penetrating peptides (CPPs) can rapidly cross plasma membranes. This study is aimed at identifying an efficient and stable CPP-SOD1 and investigating its effects on CP-induced nephrotoxicity. We recombined SOD1 with 14 different CPPs and purified them using an NTA-Ni2+ column. In in vitro experiments, CPPs-SOD1 cell membrane penetration ability and JNK/p38 MAPK signaling pathway were evaluated using Western blotting. ROS production, mitochondrial membrane potential (MMP), and cell apoptosis were determined using flow cytometry and immunofluorescence staining in VERO and HK-2 cells. For in vivo experiments, mice were administered PSF-SOD1 for 2 h before cotreatment with a single CP injection for an additional 4 days. Blood and kidney samples were collected for renal function assessment (creatinine, urea nitrogen, histopathology, TUNEL assay, and JNK/p38 MAPK signaling pathway). Compared with TAT-SOD1, we found that PSF-SOD1 is more efficient at crossing the cell membrane and is stable after transduction into cells. Pretreatment with PSF-SOD1 inhibited CP-induced apoptosis, ROS generation, and JNK/p38 MAPK activation and restored CP-induced MMP loss in VERO and HK-2 kidney cells. Treatment of mice with PSF-SOD1 inhibited CP-induced serum creatinine, blood urea nitrogen elevation, and JNK/p38 MAPK activation. H&E staining and TUNEL assay indicated that kidney tissue damage was alleviated following PSF-SOD1 pretreatment. Overall, PSF-SOD1 ameliorated CP-induced renal damage by partially reducing oxidative stress and cell apoptosis by regulating JNK/p38 MAPK signaling pathway and might be a better cytoprotective agent than TAT-SOD1.

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TUNEL assay and SCSA determine different aspects of sperm DNA damage

Andrologia ◽

10.1111/j.1439-0272.2009.01002.x ◽

2010 ◽

Vol 42 (5) ◽

pp. 305-313 ◽

Cited By ~ 64

Author(s):

R. Henkel ◽

C. F. Hoogendijk ◽

P. J. D. Bouic ◽

T. F. Kruger

Keyword(s):

Dna Damage ◽

Tunel Assay ◽

Sperm Dna Damage ◽

Sperm Dna

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Correlation between DNA synthesis in the second, third and fourth generations of spermatogonia and the occurrence of apoptosis in both spermatogonia and spermatocytes

Reproduction ◽

10.1530/rep.0.1260661 ◽

2003 ◽

pp. 661-668 ◽

Cited By ~ 15

Author(s):

J Blanco-Rodriguez ◽

C Martinez-Garcia ◽

A Porras

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Dna Synthesis ◽

Functional Significance ◽

Tunel Assay ◽

Seminiferous Epithelium ◽

Cell Cycle Checkpoints ◽

Clear Correlation ◽

Cellular Events ◽

Dna Strand

In the seminiferous epithelium, both DNA synthesis and apoptosis occur at equivalent stages in various species, with apoptosis taking place mainly at the same stages as DNA replication in the second, third and fourth spermatogonial generations. As preservation of the cellular associations found at these stages may have some functional significance, it is important to determine whether there is a correlation between these cellular events. In this study, pairs of immunoperoxidase-stained adjacent testis sections from rats, mice, rabbits and cats in which either bromodeoxyuridine incorporated into the newly synthesized DNA strand (BrdU labelling) or DNA 3' end labelling of the apoptotic DNA fragments (TUNEL assay) were detected were compared. In addition, both events were analysed in double-labelled sections. These two methods revealed a clear correlation between the occurrence of DNA replication in the second to fourth generations of spermatogonia and most physiological apoptosis taking place in both spermatogonia and spermatocytes in the three different mammalian orders (Rodentia, Lagomorpha and Carnivora). This correlation may result from the synchronization of mitotic spermatogonial and meiotic spermatocyte cell cycle checkpoints operating at these stages.

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