Springer protocols feed by Cell Biology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Selective Transport of Cationized Fluorescent Topoisomerase into Nuclei of Live Cells for DNA Damage Studies
The targeted delivery of fluorescently labeled, DNA-modifying proteins into cellular nuclei permits investigation of DNA damage and chromatin function in living cells. Commercially available protein delivery vectors cannot provide selective intranuclear transportation and primarily unload their cargo in the cytoplasm. Here we describe a simple approach for specific intranuclear transportation of vaccinia topoisomerase protein based on its cationization. The delivered protein can be observed and monitored by fluorescence microscopy. The technique is cost-efficient and time-saving. It can be useful in live cell studies. (Sou...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Selective Detection of Phagocytic Phase of Apoptosis in Fixed Tissue Sections
Degradation of apoptotic cells is finalized during the phagocytic waste-management phase of apoptosis. This eliminates genetic material present in dying cells which often contain pathological, viral, or cancerous DNA. In the waste-management phase, chromatin of apoptotic cells is engulfed and digested by professional phagocytes or surrounding tissue cells. This process is critical for the efficient completion of apoptosis and its detection is necessary in research on cancer and autoimmune diseases where clearance of dying cells plays the central role. Here we present a rapid and simple fluorescence technique for visualizat...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Atomic Force Microscopy for Analyzing Metaphase Chromosomes: Comparison of AFM Images with Fluorescence Labeling Images of Banding Patterns
The combined use of fluorescence microscopy with atomic force microscopy (AFM) has been introduced to analyze the replication-banding patterns of human chromosomes. Human lymphocytes synchronized with excess thymidine are treated with 5-ethynyl-2′-deoxyuridine (EdU) during the late S phase. EdU-labeled DNA is detected in metaphase chromosomes using Alexa Fluor 488® azide, through the 1,3-dipolar cycloaddition reaction of organic azides with the terminal acetylene group of EdU. Chromosomes with EdU incorporated during the late S phase show a banding pattern similar to the G-banding pattern of normal human chromoso...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Ultrastructural and Immunofluorescent Methods for the Study of the XY Body as a Biomarker
Structural and immunohistochemical methods have been extremely useful for the characterization of the XY body (the structure formed by the XY pair during meiotic prophase) in Man and in other mammals. These methods are widely used at the present time for the detection of abnormalities leading to human infertility. The basic ultrastructural methods are spreading of pachytene spermatocytes, thin-sectioning techniques with or without 3-D reconstructions, and the monitoring of all specimens with semi-thin sections. Immunofluorescent techniques also use spreading of meiotic cells for the analysis of the XY body, and they can be...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
FISH Methods in Cytogenetic Studies
This chapter describes the various methods derived from the protocol of standard fluorescent in situ hybridization (FISH) that are used in human, animal, plant, and microbial studies. These powerful techniques allow us to detect and physically map on interphase nuclei, chromatin fibers, or metaphase chromosomes probes derived from single-copy genes to repetitive DNA sequences. Other variants of the technique enable the co-localization of genes and the overall comparison of the genome among individuals of the same species or of different taxa. A further variant detects and localizes bacteria on tissues and cells. Overall, t...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Detection and Measurement of Necrosis in Plants
Necrosis plays a fundamental role in plant physiology and pathology. When plants or plant cell cultures are subjected to abiotic stress they initiate rapid cell death with necrotic morphology. Likewise, when plants are attacked by pathogens, they develop necrotic lesions, the reaction known as hypersensitive response. Great advances in the understanding of signaling pathways that lead to necrosis during plant–pathogen interaction have been made in the last two decades using Arabidopsis thaliana as a model plant. Further understanding of these signaling pathways, as well as those regulating the execution phase of necr...
Source: Springer protocols feed by Cell Biology - June 5, 2013 Category: Cytology Source Type: news
Use of Necrotic Markers in the Drosophila Ovary
Necrosis is a form of cell death characterized by cytoplasmic and organelle swelling, compromised membrane integrity, intracellular acidification, and increased levels of reactive oxygen species (ROS) and cytosolic Ca2+. In the Drosophila ovary, two distinct forms of cell death occur naturally. In response to starvation, caspase-dependent cell death occurs during mid-oogenesis. Additionally, the nurse cells, which support the developing oocyte, undergo developmental programmed cell death during late oogenesis after they dump their contents into the oocyte. Evidence suggests that necrosis may be playing an important ro...
Source: Springer protocols feed by Cell Biology - June 5, 2013 Category: Cytology Source Type: news
Modeling Calcium-Overload Mediated Necrosis in Drosophila
Necrotic cell death (necrosis) occurs in many acute-onset diseases. However, our poor understanding of its mechanism has greatly limited medical interventions. Here we describe two methods to establish necrosis models in Drosophila. Our strategy is to overload calcium by expression of leaky cation channels. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - June 5, 2013 Category: Cytology Source Type: news
Monitoring the Clearance of Apoptotic and Necrotic Cells in the Nematode Caenorhabditis elegans
The nematode Caenorhabditis elegans is an excellent model organism for studying the mechanisms controlling cell death, including apoptosis, a cell suicide event, and necrosis, pathological cell deaths caused by environmental insults or genetic alterations. C. elegans has also been established as a model for understanding how dying cells are cleared from animal bodies. In particular, the transparent nature of worm bodies and eggshells make C. elegans particularly amenable for live-cell microscopy. Here we describe methods for identifying apoptotic and necrotic cells in living C. elegans embryos, larvae, and adults and ...
Source: Springer protocols feed by Cell Biology - June 5, 2013 Category: Cytology Source Type: news
Necrosis in C. elegans
We present a summary of two contrasting models for studying necrosis in C. elegans and outline the methods for scoring necrosis in each. These methods are useful for the study of necrosis under other conditions in C. elegans and for comparative studies both between established and new necrosis models. We also present a list of the genetic and drug tools available for perturbing pathways known to be important for initiation or execution of necrosis and describe their use in C. elegans. Specifically, we outline methods to inhibit autophagy, to perturb calcium homeostasis, and to disrupt lysosomal function in the C. elegans s...
Source: Springer protocols feed by Cell Biology - June 5, 2013 Category: Cytology Source Type: news
Quantification of Genetically Controlled Cell Death in Budding Yeast
Yeast are the foremost genetic model system. With relative ease, entire chemical libraries can be screened for effects on essentially every gene in the yeast genome. Until recently, researchers focused only on whether yeast were killed by the conditions applied, irrespective of the mechanisms by which they died. In contrast, considerable effort has been devoted to understanding the mechanisms of mammalian cell death. However, most of the methodologies for detecting programmed apoptotic and necrotic death of mammalian cells have not been applicable to yeast. Therefore, we developed a cell death assay for baker’s yeast...
Source: Springer protocols feed by Cell Biology - June 5, 2013 Category: Cytology Source Type: news
Identification and Characterization of Programmed Cell Death Markers in Bacterial Models
In eukaryotic organisms facing terminal stress, activation of genetically encoded cell death pathways underlies fundamental changes in core cellular processes and functional modification of critical biomolecules. These physiological alterations manifest themselves as phenotypic hallmarks during programmed cell death, and are markers of the particular mode of death initiated. A growing volume of work has illustrated that prokaryotes too are capable of exhibiting hallmarks of programmed cell death, albeit without the multiple, tight regulatory layers which control these events in higher order organisms. (Source: Springer pro...
Source: Springer protocols feed by Cell Biology - June 5, 2013 Category: Cytology Source Type: news
Quantitation of Acute Necrosis After Experimental Myocardial Infarction
Myocardial infarction (MI) is death and necrosis of myocardial tissue secondary to ischemia. MI is associated with adverse cardiac remodeling, progressive heart chamber dilation, ventricular wall thinning, and loss of cardiac function. Myocardial necrosis can be experimentally induced in rodents to simulate human MI by surgical occlusion of coronary arteries. When induced in knockout or transgenic mice, this model is useful for the identification of molecular modulators of cell death, cardiac remodeling, and preclinical therapeutic potential. Herein we outline in tandem, methods for microsurgical ligation of the left anter...
Source: Springer protocols feed by Cell Biology - June 5, 2013 Category: Cytology Source Type: news
Histological Analysis of Neurodegeneration in the Mouse Brain
Neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) are characterized by chronic and progressive neuronal loss. Being able to detect and quantify neurodegeneration is the first step to identify mechanisms underlying neuronal cell death and to develop novel therapeutic strategies. In this chapter, we describe a practical method for detecting and quantifying neurodegeneration in adult and aging mouse brains based on protocols developed in our laboratory over the last decade. We include protocols on sample preparation, immunohistochemical analysis, and stereological methods for...
Source: Springer protocols feed by Cell Biology - June 5, 2013 Category: Cytology Source Type: news
Analysis of Cell Death by Electron Microscopy
Cell death is said to occur mostly by two alternative, opposite modes: apoptosis, which involves a highly genetically regulated and elaborate network of biochemical events and cascades, and necrosis, considered a passive cell death without underlying regulatory mechanisms. Here, we describe the different morphological features of cells undergoing apoptotic and necrotic cell death, through the analysis of transmission (TEM) and scanning (SEM) electron microscopy. TEM allows detailed studies of ultrastructural changes, within the cell, such as the nuclear alteration, the cytoplasmic reorganization, and the loss of membrane i...
Source: Springer protocols feed by Cell Biology - June 5, 2013 Category: Cytology Source Type: news
