scholarly journals TUNEL Apoptotic Cell Detection in Tissue Sections: Critical Evaluation and Improvement

1998 ◽  
Vol 46 (3) ◽  
pp. 327-334 ◽  
Author(s):  
Françoise Labat-Moleur ◽  
Christiane Guillermet ◽  
Philippe Lorimier ◽  
Catherine Robert ◽  
Sylvie Lantuejoul ◽  
...  
Keyword(s):  
Ph Value ◽  
Apoptotic Cell ◽  
Critical Evaluation ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Cell Detection ◽  
Apoptotic Cells ◽  
Intense Staining ◽  
Tissue Sections ◽  
Enhanced Sensitivity ◽  
Final Answer

TUNEL, i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, has become a widely used staining method to assist in detection of apoptotic cells in tissue sections. However, despite its apparent simplicity, this technique has led to considerable disappointment because of its serious limitations in sensitivity and, even more, in specificity. We reviewed the limitations and artifacts of TUNEL and designed a comprehensive protocol to reassess the various procedures in use for five crosslinking and/or precipitating fixatives. By introducing microwave heating in extreme pH-value solutions (pH 3 for formalin and pH 10.6 for Bouin's fixative) coupled with proteolysis, we obtained an intense staining of 70–80% of apoptotic cells and bodies on archival tissue blocks, with little or no background. Owing to the enhanced sensitivity, early stages of apoptosis could be visualized and may enlarge our vision of the apoptotic cell beyond the mere image of shrinkage necrosis. We conclude that TUNEL remains a technique as useful as it is delicate, requiring critical interpretation of the staining. This study points out that, on archival tissues, despite the technical improvements we propose no protocol can be the final answer to all problems. Technique must be readjusted for any variation in tissue processing. However, step-by-step progress has rendered this method not only applicable but also performable within the constraints of archival surgical pathology specimens.

scholarly journals 114 PORCINE EMBRYO FRAGMENTATION, DEVELOPMENT AND APOPTOSIS: A CONFOCAL MICROSCOPY STUDY

2005 ◽  
Vol 17 (2) ◽  
pp. 207
Author(s):  
B. Mateusen ◽  
A. Van Soom ◽  
D. Maes ◽  
A. de Kruif
Keyword(s):  
Confocal Microscopy ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Apoptotic Cells ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Developmental Arrest ◽  
Morula Stage ◽  
Embryonic Arrest

The relationship between embryonic fragmentation, embryonic arrest, and apoptosis has been the subject of some controversy (Hardy K 1999 Rev. Reprod. 4, 125–134). In order to investigate possible links, in vivo-produced, in vitro-cultured porcine embryos (n = 132) were scored for developmental stage and fragmentation at 7 days post insemination (dpi) and processed for propidium iodide and annexin V labelling. After fixation, embryos were processed for terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL). Using confocal microscopy, a cell was categorized apoptotic if (i) it had a fragmented or condensed nucleus, (ii) the cell membrane was annexin V-positive, and (iii) the nucleus was TUNEL labelled. An apoptotic cell ratio (ACR) was determined as the percentage of apoptotic cells per embryo. Differences in the % of fragmented and apoptotic embryos and correlations were analyzed using chi-square. Logistic regression was used to compare the average fragmentation % and the ACR. Sixty-one embryos (46%) arrested during the culture period, with 8 embryos arresting before or at the 4-cell stage. Significantly more arrested embryos were fragmented compared to embryos that were blastocysts at 7 dpi. Also, the average fragmentation percentage was significantly higher for arrested embryos compared to blastocysts. The correlation detected between developmental arrest and fragmentation was 0.60 (P < 0.05). None of the embryos without fragmentation had cells categorized as apoptotic, whereas 50 out of 55 embryos with fragmentation possessed apoptotic cells, which led to a correlation of 0.87 (P < 0.01) between fragmentation and apoptosis. The percentage of embryos with apoptotic cells was significantly higher for embryos arrested during the 5-cell to the morula stage compared to embryos that arrested before or at the 4-cell stage and embryos with blastocyst development at 7 dpi. The average ACR of embryos arrested during the 5-cell to the morula stage was significantly higher compared to the average ACR of blastocysts at 7 dpi. The correlation detected between the developmental arrest, during the 5-cell to the morula stage period and apoptosis was 0.57 (P < 0.01). Taken together, significant correlations between fragmentation, developmental arrest and apoptosis were detected. However, the association between embryonic arrest and apoptosis could be established only for embryos arrested after embryonic genome activation.

Induction of secondary apoptosis, inflammation, and lung fibrosis after intratracheal instillation of apoptotic cells in rats

2006 ◽  
Vol 290 (4) ◽  
pp. L695-L702 ◽  
Author(s):  
Liying Wang ◽  
James F. Scabilloni ◽  
James M. Antonini ◽  
Yon Rojanasakul ◽  
Vincent Castranova ◽  
...  
Keyword(s):  
Cell Apoptosis ◽  
Apoptotic Cell ◽  
Death Receptor ◽  
Intratracheal Instillation ◽  
Inflammatory Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Apoptotic Cells ◽  
Lung Pathology ◽  
Continuous Induction

Uncontrolled apoptosis has been associated with several pulmonary disorders; however, the molecular mechanism underlying this process and the fate of apoptotic cells in vivo are unclear. Here we show that direct administration of apoptotic cells to the lungs of rats caused pulmonary inflammation and fibrosis, as indicated by emigration of inflammatory cells to the air spaces, TNF-α immunoreactivity, and connective tissue accumulation, indicating a direct relationship between apoptotic cells and the observed lung pathologies. To determine how the lungs process the accumulated apoptotic cells, normal or apoptotic cells from autologous donor rats were labeled with fluorescent nanobeads and intratracheally instilled into the lungs of rats. Probe distribution and lung cell apoptosis were determined at various times over a 28-day period by confocal fluorescence microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, respectively. Labeled apoptotic cells were cleared by lung macrophages within 1 wk after the treatment. However, the total number of apoptotic cells in the lung remained high at 28 days posttreatment. The results indicate a continuous induction of secondary apoptosis by apoptotic cell instillation, which may contribute to the observed lung pathology. Analysis of lung cell apoptosis by caspase assays showed an elevation of caspase-8 but not caspase-9 in the treatment group at 28 days posttreatment, indicating involvement of the death receptor-mediated pathway in the apoptotic process. Together, our results demonstrate a direct effect of apoptotic cell accumulation on inflammatory and fibrotic pulmonary responses and the continuous induction of lung cell apoptosis by apoptotic cell instillation.

Changqin NO. 1 inhibits neuronal apoptosis via suppressing GAS5 expression in a traumatic brain injury mice model

2019 ◽  
Vol 400 (6) ◽  
pp. 753-763 ◽  
Author(s):  
Xingping Dai ◽  
Min Yi ◽  
Dongsheng Wang ◽  
Yanyi Chen ◽  
Xia Xu
Keyword(s):  
Traumatic Brain Injury ◽  
Brain Injury ◽  
Neuronal Apoptosis ◽  
Apoptotic Cell ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Luciferase Reporter ◽  
Cell Detection ◽  
Neuroprotective Effects ◽  
Newborn Mice

Abstract The present study was designed to investigate the mechanism of the traditional Chinese medicine Changqin NO. 1 on the amelioration of traumatic brain injury (TBI). Adult male C57BL/6J mice and newborn mice were used to generate a mouse TBI model and harvest primary neurons, respectively. The localizations of specific neural markers neuropilin-1 (Nrp-1), growth-associated protein-43 (GAP-43) and microtubule-associated protein Tau (Tau) were examined in brain tissues by immunohistochemistry. Terminal deoxynucleotidyl transferase dUTP nick end labeling apoptotic cell detection in tissue sections and the CCK-8 cell viability assay were performed to examine neuronal apoptosis. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were also carried out in this study. The association between long non-coding RNA (lncRNA) growth-arrest specific 5 (GAS5), miR-335 and RAS p21 GTPase activating protein 1 (Rasa1) was disclosed using the dual-luciferase reporter assay. Changqin NO. 1 inhibited TBI-induced neuronal apoptosis in vivo and in vitro. GAS5 functioned as a competing endogenous RNA (ceRNA) by sponging miR-335 to upregulate Rasa1 expression in mouse neuronal cells. Further investigations demonstrated that GAS5 promoted neuronal apoptosis following TBI via the miR-335/Rasa1 axis. In vivo experiments indicated that Changqin NO. 1 exerted neuroprotection during TBI via the GAS5/miR-335/Rasa1 axis. Changqin NO. 1 promoted neuroprotective effects by inhibiting neuronal apoptosis via the GAS5/miR-335/Rasa1 axis in TBI.

scholarly journals The inflammatory response induced by Pseudomonas aeruginosa in macrophages enhances apoptotic cell removal

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Adriana Valeria Jäger ◽  
Paula Arias ◽  
Maria Virginia Tribulatti ◽  
Marcela Adriana Brocco ◽  
Maria Victoria Pepe ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Pseudomonas Aeruginosa ◽  
Inflammatory Response ◽  
Apoptotic Cell ◽  
Bacterial Pathogen ◽  
Environmental Cues ◽  
Apoptotic Cells ◽  
Phagocytic Function ◽  
Inflammatory Stimulus ◽  
Inflammatory Conditions

AbstractPathogens phagocytosis and the uptake of apoptotic cells (efferocytosis) are essential macrophages tasks, classically considered as mutually exclusive. Macrophages have been observed to polarize into either pro-inflammatory/microbicidal or anti-inflammatory/efferocytic phenotypes. However, macrophage functions have shown to be more complex. Furthermore, little is known about the regulation of efferocytosis under inflammatory conditions. In this study, we elucidate the modulation of the macrophage efferocytic function during an inflammatory stimulus. We find that bone marrow-derived macrophages (BMDM) are very efficient in engulfing both the bacterial pathogen Pseudomonas aeruginosa and apoptotic cells. BMDM showed a high bactericidal capacity unaffected by the concomitant presence of apoptotic material. Plasticity in macrophage programming, in response to changing environmental cues, may modulate efferocytic capability. In this work, we further show that, after phagocyting and processing Pseudomonas aeruginosa, macrophages highly increase their efferocytic capacity without affecting their phagocytic function. Moreover, we demonstrate that Pseudomonas aeruginosa enhances efferocytosis of these phagocytes through the IL-6 signaling pathway. Our results show that the inflammatory response generated by the bacterial processing enhances these macrophages’ capacity to control inflammation through an increased efferocytosis.

scholarly journals Cytotoxicity, Antiproliferative Effects, and Apoptosis Induction of Methanolic Extract ofCynometra caulifloraLinn. Whole Fruit on Human Promyelocytic Leukemia HL-60 Cells

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
T-Johari S. A. Tajudin ◽  
Nashriyah Mat ◽  
Abu Bakar Siti-Aishah ◽  
A. Aziz M. Yusran ◽  
Afnani Alwi ◽  
...  
Keyword(s):  
Cell Death ◽  
Methanolic Extract ◽  
Apoptotic Cell ◽  
Mouse Fibroblast ◽  
Promyelocytic Leukemia ◽  
Apoptotic Cell Death ◽  
Apoptotic Cells ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Methanolic extract ofCynometra cauliflorawhole fruit was assayed for cytotoxicity against the human promyelocytic leukemia HL-60 and the normal mouse fibroblast NIH/3T3 cell lines by using the MTT assay. The CD50of the extract for 72 hours was 0.9 μg/mL whereas the value for the cytotoxic drug vincristine was 0.2 μg/mL. The viability of the NIH/3T3 cells was at 80.0% when treated at 15.0 μg/mL. The extract inhibited HL-60 cell proliferation with dose dependence. AO/PI staining of HL-60 cells treated with the extract revealed that majority of cells were in the apoptotic cell death mode. Flow cytometry analysis of HL-60 cells treated at CD50of the extract showed that the early apoptotic cells were 31.0, 26.3 and 19.9% at 24, 48, and 72 hours treatment, respectively. The percentage of late apoptotic cells was increased from 62.0 at 24 hours to 64.1 and 70.2 at 48 and 72 hours, respectively. Meanwhile, percent of necrotic cells were 4.9, 6.6, and 8.5 at 24, 48, and 72 hours, respectively. This study has shown that the methanolic extract ofC. cauliflorawhole fruit was cytotoxic towards HL-60 cells and induced the cells into apoptotic cell death mode, but less cytotoxic towards NIH/3T3 cells.

scholarly journals Hydrocortisone decreases apoptosis in jejunum of horses subjected to experimental ischemia and reperfusion

2011 ◽  
Vol 31 (6) ◽  
pp. 471-476 ◽  
Author(s):  
Geraldo Eleno S. Alves ◽  
Heloisa M.F. Mendes ◽  
Tiago G.S. Alves ◽  
Rafael R. Faleiros ◽  
Anilton C. Vasconcelos ◽  
...  
Keyword(s):  
Cell Death ◽  
Time Course ◽  
Apoptotic Cell ◽  
Treated Group ◽  
Apoptotic Cell Death ◽  
Apoptotic Cells ◽  
Ischemia And Reperfusion ◽  
Beneficial Effects ◽  
Time Zero ◽  
Venous Ischemia

In order to evaluate the effect of hydrocortisone on apoptosis in the jejunum of horses subjected to ischemia and reperfusion, ten horses were paired and grouped into two groups - treated (n=5) and non treated (n=5). Segments of the jejunum were used as controls (C), or as venous ischemia (VIsc), which were subjected to 2h of ischemia followed by 2 or 12h of reperfusion. C samples were collected at time zero (prior to ischemia) and VIsc samples were collected at 2h of ischemia and at 2 and 12h of reperfusion. TUNEL positive apoptotic cells were counted in 10 microscopical fields in deep mucosa from each horse throughout the time course. After 12h of reperfusion, the number of apoptotic cells in treated group were significantly lower than in untreated animals, indicating that hydrocortisone inhibits apoptosis. These results indicate that hydrocortisone has a beneficial effects favoring the maintenance of jejunal integrity in horses with ischemia and reperfusion injuries by preventing apoptotic cell death.

scholarly journals Different Roles of a Rat Cortical Thymic Epithelial Cell LineIn Vitroon Thymocytes and Thymocyte Hybridoma Cells: Phagocytosis, Induction of Apoptosis, Nursing and Growth Promoting Activities

2002 ◽  
Vol 9 (2) ◽  
pp. 63-72 ◽  
Author(s):  
Dragana Vucevic ◽  
Miodrag Colic ◽  
Petar Popovic ◽  
Sonja Gašic
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Apoptotic Cell ◽  
Hybridoma Cells ◽  
Thymic Epithelial Cell ◽  
Apoptotic Cells ◽  
Th Cells ◽  
Nursing Activity ◽  
Induction Of Apoptosis ◽  
Stimulation Of

In this work, the interaction between a rat cortical thymic epithelial cell (TEC) line (R-TNC.1) with nursing activity and thymocytes as well as BWRT 8 thymocyte hybridoma (TH) cells has been studied. The R-TNC.1 cell line significantly bound thymocytes and TH. Binding was stronger during the first 30 min of cell incubation and was followed by a progressive deadhesion. Among adherent thymocytes the proportion of apoptotic cells increased with culture time which was a consequence of higher capacity of the line for binding of apoptotic than viable cells and induction of apoptosis in a subset of adherent thymocytes. Emperiopolesis activity of this thymic nurse cell (TNC) line was manifested by engulfment of thymocytes as well as TH cells. A subset of viable intra-TNC thymocytes has been triggered to die by apoptosis, whereas other internalized thymocytes have been stimulated to proliferate, as measured by an increase in the percentage of cells in mitosis and higher incorporation of bromodeoxyuridine (BrdU), in comparison to thymocytes cultivated alone. A significant stimulation of proliferation of engulfed TH cells was also observed. The R-TNC.1 cell line efficiently phagocytosed both apoptotic thymocytes and TH, and the process is followed by intra-TNC destruction of ingested cells. Cumulatively, these results suggest different role of the R-TNC.1 clone: phagocytosis of apoptotic cells; induction of apoptotic cell death in a subset of both bound and internalized thymocytes and stimulation of proliferation of a subset of intra-TNC thymocytes or TH cells.

scholarly journals A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA.

1993 ◽  
Vol 41 (1) ◽  
pp. 7-12 ◽  
Author(s):  
J H Wijsman ◽  
R R Jonker ◽  
R Keijzer ◽  
C J van de Velde ◽  
C J Cornelisse ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Staining Method ◽  
Morphological Characteristics ◽  
Image Cytometry ◽  
Apoptotic Cells ◽  
Dna Breaks ◽  
Paraffin Sections ◽  
Tissue Sections

Apoptosis (programmed cell death) can be difficult to detect in routine histological sections. Since extensive DNA fragmentation is an important characteristic of this process, visualization of DNA breaks could greatly facilitate the identification of apoptotic cells. We describe a new staining method for formalin-fixed, paraffin-embedded tissue sections that involves an in situ end-labeling (ISEL) procedure. After protease treatment to permeate the tissue sections, biotinylated nucleotides are in situ incorporated into DNA breaks by polymerase and subsequently stained with DAB via peroxidase-conjugated avidin. Staining of cells with the morphological characteristics of apoptosis was demonstrated in tissues known to exhibit programmed cell death, i.e., prostate and uterus after castration, tumors, lymph node follicles, and embryos. Apoptotic cells could be discriminated morphologically from areas of labeled necrotic cells, in which DNA degradation also occurs. Because apoptosis is relatively easily recognized in H&E-stained sections of involuting prostates of castrated rats, we used this model system to validate the ISEL method for the quantification of apoptotic cells. A high correlation was found between the fractions of ISEL-labeled cells and the fractions of apoptotic cells that were morphologically determined in adjacent sections. We conclude that ISEL is a useful technique for quantification of apoptosis in paraffin sections, especially for those tissues in which morphological determination is difficult. Furthermore, this new staining method enables the use of automated image cytometry for evaluating apoptosis.

scholarly journals Mitomycin C Induces Apoptosis in Rheumatoid Arthritis Fibroblast-Like Synoviocytes via a Mitochondrial-Mediated Pathway

2015 ◽  
Vol 35 (3) ◽  
pp. 1125-1136 ◽  
Author(s):  
Chuqi Yan ◽  
Dechao Kong ◽  
Dong Ge ◽  
Yanming Zhang ◽  
Xishan Zhang ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Viability ◽  
Mitomycin C ◽  
Apoptotic Cell ◽  
Chronic Inflammatory Disease ◽  
Cell Counting ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Control Treatment ◽  
Induced Apoptosis ◽  
Fibroblast Like Synoviocytes

Background/Aims: Rheumatoid arthritis (RA) is a systemic chronic inflammatory disease characterised by prominent synoviocyte hyperplasia and a potential imbalance between the growth and death of fibroblast-like synoviocytes (FLS). Mitomycin C (MMC) has previously been demonstrated to inhibit fibroblast proliferation and to induce fibroblast apoptosis. However, the effects of MMC on the proliferation and apoptosis of human RA FLS and the potential mechanisms underlying its effects remain unknown. Methods: Cell viability was determined using the Cell Counting Kit-8 assay. Apoptotic cell death was analysed via Annexin V-FITC/PI double staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling. The production of intracellular reactive oxygen species (ROS) was assessed via flow cytometry, and the changes in mitochondrial membrane potential (ΔΨm) were visualized based on JC-1 staining via fluorescence microscopy. The expression of apoptosis-related proteins was determined via Western blot. Results: Treatment with MMC significantly reduced cell viability and induced apoptosis in RA FLS. Furthermore, MMC exposure was found to stimulate the production of ROS and to disrupt the ΔΨm compared to the control treatment. Moreover, MMC increased the release of mitochondrial cytochrome c, the ratio of Bax/Bcl-2, the activation of caspase-9 and caspase-3, and the subsequent cleavage of poly(ADP-ribose) polymerase. Conclusion: Our findings suggest that MMC inhibits cell proliferation and induces apoptosis in RA FLS, and the mechanism underlying this MMC-induced apoptosis may involve a mitochondrial signalling pathway.

Efficient migration of dendritic cells toward lymph node chemokines and induction of TH1 responses require maturation stimulus and apoptotic cell interaction

Blood ◽  
2005 ◽  
Vol 106 (5) ◽  
pp. 1734-1741 ◽  
Author(s):  
Nicolas Bertho ◽  
Henri Adamski ◽  
Louis Toujas ◽  
Martine Debove ◽  
Jean Davoust ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Lymph Node ◽  
Immune Responses ◽  
Tumor Cells ◽  
Apoptotic Cell ◽  
Apoptotic Cells ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Tnf Α

Abstract Dendritic cells (DCs) have the unique ability to initiate primary immune responses, and they can be conditioned for vaccinal purposes to present antigens after the engulfment of apoptotic cells. To recruit the rare antigen-specific naive T cells, DCs require a maturation step and subsequent transport toward lymph node (LN). To date, prostaglandin E2 (PGE2) is the best-characterized compound inducing this LN-directed migration in vitro, but PGE2 may skew the immune responses in a TH2 direction. We demonstrate here that on incubation with apoptotic tumor cells and tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS), human monocyte-derived DCs become fully mature and acquire high migratory capacities toward LN-directing chemokines. The migration of TNF-α-treated DCs occurs only after cotreatment with apoptotic cells but not with necrotic cells. DC migration requires CD36 expression and incubation with apoptotic cells in the presence of heat-labile serum components. Moreover, on treatment with apoptotic cells and LPS, the migrating DCs are able to recruit naive T cells to generate TH1 immune responses. Our results show that the cotreatment of DCs with apoptotic tumor cells and inflammatory signals is promising for the design of an antitumoral DC-based vaccine. (Blood. 2005;106:1734-1741)

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