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Detection of Apoptosis in the Central Nervous System

Apoptosis occurs in the nervous system during normal development, but can also be induced by disease or after exogenous insults such as DNA damage. Depending on the magnitude and timing of the stimulus, apoptosis can be sporadic or widespread. Because of the highly ordered structure of the nervo ...

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Detection of Apoptosis in Mammalian Development

Mammalian development is dependent on an intricate orchestration of cell proliferation and death. Deregulation in the levels, localization, and type of cell death can lead to disease and even death of the developing embryo. The mechanisms involved in such deregulation are many; alterat ...

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Assessment of Apoptotic Cell Phagocytosis by Macrophages

Cells undergo apoptosis during development, tissue homeostasis, and disease, and are rapidly cleared by both professional and nonprofessional phagocytes. In the whole animal, this process is remarkably efficient and usually goes unnoticed. It is estimated that 2 � 1011 cells are clear ...

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Multiple Approach to Analyzing the Role of MicroRNAs in Apoptosis

MicroRNAs (miRNAs) are noncoding RNAs whose hallmarks are the very short sequences and the ability to repress the translation and/or transcription of target genes. miRNAs can have diverse functions, including regulation of cellular differentiation, proliferation, and embryog ...

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Detection of Uncoupling Protein-2 (UCP2) As a Mitochondrial Modulator of Apoptosis

There is an increasing evidence that uncoupling protein-2 (UCP2), a recently identified molecular sensor and suppressor of mitochondrial reactive oxygen species (ROS), plays an important role in �regulating apoptosis in different cell systems. A great technical difficulty that m ...

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Regulation of Apoptosis by the Unfolded Protein Response

In eukaryotic cells, the endoplasmic reticulum (ER) serves many specialized functions including bio-synthesis and assembly of membrane and secretory proteins, calcium storage and production of lipids and sterols. As a plant for protein folding and posttranslational modifica ...

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Fluorometric Methods for Detection of Mitochondrial Membrane Permeabilization in Apoptosis

The mitochondrial regulation of cell death involves the release of proapoptotic factors, such as cytochrome c, Smac-DIABLO, AIF, OMI/HtrA2, by disruption of the outer mitochondrial membrane (OMM) permeability barrier that is controlled by pro- and antiapoptotic proteins of the Bcl-2 f ...

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Measurement of Changes in Cdk2 and Cyclin O-Associated Kinase Activity in Apoptosis

Many cell cycle regulatory proteins have been shown to be able to regulate cell death. Activation of Cdk2 has been shown to be necessary for apoptosis of quiescent cells such as thymocytes, neurons, and endothelial cells. This activation is stimulus-specific because it occurs in glucocorti ...

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Assays to Measure p53-Dependent and -Independent Apoptosis

Paramount to the maintenance of normal tissue homeostasis is the induction of programmed cell death, otherwise known as apoptosis. Several disease states, including cancer, are characterized by an inability to remove unwanted cells due to a failure to commit to apoptosis. What is more, apop ...

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Application of In Vivo EPR for Tissue pO2 and Redox Measurements

The technique of electron paramagnetic resonance (EPR) spectroscopy is more than 50 years old, but only recently it has been used for in vivo studies. Its limited application in the past was due to the problem of high nonresonant dielectric loss of the exciting frequency because of high water conte ...

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Detection of Cell Death by Autophagy

Autophagy (Greek: Self digestion) is an intracellular process involved in removal of damaged or misfolded proteins or organelles. Damaged or misfolded proteins or organelles are first engulfed in a membraneous structure called autophagosome, and then the autophagosome fuse with l ...

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Methods to Analyze S-nitrosylation of Proteins Involved in Apoptosis

Nitric oxide (NO) is an important signaling molecule that plays a key role in various physiological and pathological processes. One of the well-established mechanisms by which NO regulates the function of various target proteins is through S-nitrosylation. NO readily reacts with thiol ...

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Methods to Analyze Transglutamination of Proteins Involved in Apoptosis

Enhanced expression of transglutaminases is a frequent, though not obligatory phenomenon in apoptosis, which is associated with cells dying in steady interaction with their tissue environment. Modification of cellular proteins by transglutamination is a tightly controlled ...

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Methods to Analyze Cellular Necroptosis

Necroptosis is a mechanism of necrotic cell death induced by external stimuli in the form of death domain receptor (DR) engagement by their respective ligands, TNF-alpha, Fas ligand (FasL) and TRAIL, under conditions when apoptotic cell death execution is prevented, e.g. by caspase inhibit ...

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Detection of Apoptosis in Cell-Free Systems

Apoptosis is a fundamental process required for proper embryonic development. Various methods have been described to detect apoptosis both in vitro as well as in vivo. Activation of caspases represents the key event in the apoptotic process. To dissect the molecular events leading to casp ...

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Detection of Apoptosis in Tissue Sections

TUNEL-based assays were used to demonstrate the presence of apoptotic cells in tissue sections derived from target tissues of animal models of different diseases. Emphasis was placed on tissue preparation and fixation, as these are crucial to successful histological staining. The pro ...

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Live to Dead Cell Imaging

Live cell imaging allows several key apoptotic events to be visualized in a single cell over time. These include mitochondrial outer membrane permeabilization (MOMP), mitochondrial dysfunction, phosphatidylserine exposure, and membrane permeabilization. Here we describe a ...

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Flow Cytometry-Based Apoptosis Detection

An apoptosing cell demonstrates multitude of characteristic morphological and biochemical features, which vary depending on the stimuli and the cell type. The gross majority of classical apoptotic hallmarks can be rapidly examined by flow and image cytometry. Cytometry thus beca ...

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Analysing Caspase Activation and Caspase Activity in Apoptotic Cells

Apoptotic cell death is characterised by various morphological and biochemical changes. Cysteine proteases of the caspase family play key roles in the execution of apoptosis and in the maturation of proinflammatory cytokines. During apoptosis signalling, caspase precursors u ...

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Evaluating the Localization and DNA Binding Complexity of Histones in Mature Sperm

The paternal genome in many animal taxa is efficiently packaged into the sperm nucleus by switching from a histone (nucleosome)-based chromatin configuration to one using predominantly protamines. Nonetheless, various studies have shown that some nucleosomes, often containi ...

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