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.Immunohistological Tools to Discriminate Apoptotic and Necrotic Cell Death in the Skin
Perturbances in skin homeostasis are responsible for the development of skin inflammatory diseases such as psoriasis or atopic dermatitis. While the role of apoptosis has been extensively studied in the skin, the role of the newly described programmed necrosis also termed necroptosis in human skin remains poorly understood. We have recently described a mouse model of skin inflammation dependent on necroptotic cell death. Here we describe an immunohistological protocol allowing for the discrimination of apoptotic from necroptotic cell death in a single staining procedure on tissue sections. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - June 5, 2013 Category: Cytology Source Type: news
Analysis of Pyroptosis in Bacterial Infection
Eukaryotic cells undergo death by several different mechanisms: apoptosis, a cell death that prevents inflammatory response; necrosis, when the cell membrane lyses and all the intracellular content is spilled outside; and pyroptosis, a cell death that is accompanied by the release of inflammatory cytokines by the dying cells. Pyroptosis is designed to attract a nonspecific innate response to the site of infection or tumor. In this chapter, we describe the methods used to study pyroptosis in a mammalian cell. The model organism used is Mycobacterium tuberculosis, which suppresses pyroptosis by macrophages, and possibly in d...
Source: Springer protocols feed by Cell Biology - June 5, 2013 Category: Cytology Source Type: news
Methods for the Study of Entosis
Entosis is a recently described nonapoptotic cell death mechanism that is initiated by the engulfment of live epithelial cells, leading to the formation of “cell-in-cell” structures. Entotic cell engulfment is induced by matrix detachment, and is driven by imbalances in actomyosin contraction between neighboring cells. Here we describe methods to quantify the formation of cell-in-cell structures by entosis, for cells cultured in suspension or in soft agar, by fluorescence imaging and time-lapse microscopy. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - June 5, 2013 Category: Cytology Source Type: news
Fluorescent Biosensors for the Detection of HMGB1 Release
During necrosis and following some instances of apoptosis (in particular in the absence of a proficient phagocytic system), the nonhistone chromatin component high-mobility group box 1 (HMGB1) is released in the extracellular space. In vivo, extracellular HMGB1 can bind Toll-like receptor 4 on the surface of dendritic cells, de facto operating as a danger-associated molecular pattern and alarming the organism to the presence of stressful conditions. Recent results indicate that the release of HMGB1 is one of the key features for cell death to be perceived as immunogenic, i.e., to be capable of triggering a cognate immune r...
Source: Springer protocols feed by Cell Biology - June 5, 2013 Category: Cytology Source Type: news
Activity Assays for Receptor-Interacting Protein Kinase 1:A Key Regulator of Necroptosis
Necroptosis is a novel form of regulated non-apoptotic cell death, which displays morphological features of necrosis. The kinase activity of receptor-interacting protein kinase 1 (RIP1) is a critical component in signaling for necroptosis. The development of assays to evaluate RIP1 kinase activity is important in the further development of existing and novel inhibitors of necroptosis. Here, we describe RIP1 protein expression and purification from mammalian and insect cells as well as two in vitro kinase assays to detect RIP1 kinase activity and inhibition. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - June 5, 2013 Category: Cytology Source Type: news
Time-Lapse Imaging of Necrosis
The processes of dying are as tightly regulated as those of growth and proliferation. Recent work into the molecular pathways that regulate and execute cell death have uncovered a plethora of signalling cascades that lead to distinct modes of cell death, including “apoptosis,” “necrosis,” “autophagic cell death,” and “mitotic catastrophe.” Given that cells can readily switch from one form of death to another, it is vital to carefully monitor the form of death under investigation. Particularly, end-point techniques are intrinsically unsuitable for assessing apoptosis versus ne...
Source: Springer protocols feed by Cell Biology - June 5, 2013 Category: Cytology Source Type: news
Navigation to the Graveyard-Induction of Various Pathways of Necrosis and Their Classification by Flow Cytometry
Apoptosis and necrosis reflect the program of cell death employed by a dying cell and the final stage of death, respectively. Whereas apoptosis is defined as a physiological, highly organized cell death process, necrosis is commonly considered to be accidental and uncontrolled. Physiological and weak pathological death stimuli preferentially induce apoptosis, while harsh non-physiological insults often immediately instigate (primary) necrosis. If an apoptosing cell transits into a phase of plasma membrane disintegration, this stage of death is referred to as secondary or post-apoptotic necrosis. (Source: Springer protocols...
Source: Springer protocols feed by Cell Biology - June 5, 2013 Category: Cytology Source Type: news
M-Current Recording from Acute DRG Slices
Electrophysiological recordings from an acutely sliced preparation provide information on ionic currents and excitability of native neurons under near physiological conditions. Although this technique is commonly used on central nervous system structures such as spinal cord and brain, structures within the peripheral nervous system (including sensory ganglia and fibers) have proven to be much more difficult to study in acute preparations. Here we describe a method for patch-clamp recordings from rat dorsal root ganglion (DRG) slices. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Recording Dendritic Ion Channel Properties and Function from Cortical Neurons
Dendrites emerging from the cell bodies of neurons receive the majority of synaptic inputs. They possess a plethora of ion channels that are essential for the processing of these synaptic signals. To fully understand how dendritic ion channels influence neuronal information processing, various patch-clamp techniques that allow electrophysiological recordings to be made directly from dendrites have been developed. In this chapter, I describe one such method that is suitable for making electrophysiological recordings from the apical dendrites of hippocampal and cortical pyramidal neurons. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Isotope Labeling Strategies for Analysis of an Ion Channel Cytoplasmic Domain by NMR Spectroscopy
As large, multimeric, integral membrane proteins, ion channels pose technical challenges to analysis by NMR spectroscopy. Here we present a strategy to overcome some of these technical hurdles, using a representative ion channel modulatory domain, the regulator of K+ conductance (RCK) domain from a K+ channel cloned from Thermoplasma volcanium. By introducing a mutation to limit the stoichiometry of the octameric RCK domain “gating ring” complex to its dimeric building block, NMR spectral resolution can be greatly improved. Here we present protocols for efficient production of highly deuterated, uniformly 15N-l...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Analysis of Ca2+-Binding Sites in the MthK RCK Domain by X-Ray Crystallography
Regulator of K+ conductance (RCK) domains form a conserved class of ligand-binding domains that control the activity of a variety of prokaryotic and eukaryotic K+ channels. Structural analysis of these domains by X-ray crystallography has provided insight toward mechanisms underlying ligand binding and channel gating, and thus the experimental strategies aimed at determining structures of liganded and unliganded forms of the domains may be useful in analysis of other ligand-binding domains. Here, we describe a basic strategy for crystallographic analysis of the RCK domain from the MthK channel, for determination of its Ca2...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Cysteine-Based Cross-Linking Approach to Study Inter-domain Interactions in Ion Channels
Cysteine contains a highly reactive thiol group and therefore under oxidizing conditions a disulfide bond can form between a pair of cysteines that are juxtaposed in the close vicinity, which can be only reversed by reducing agents. These attributes have been elegantly exploited to study the functional role of an interaction or contact between two adjacent domains that are present in ion channels or virtually in any proteins, by introducing double cysteine substitutions at the domain interface and measuring changes in the ion channel functions arising from cross-linking the two substituted cysteines via formation of a disu...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Site-Directed Mutagenesis to Study the Structure–Function Relationships of Ion Channels
Ion channels mediate a wide variety of physiological processes by forming small pores across the membranes that allow regulated flow of ions into or out of the cell. The primary linear sequences of ion channel proteins, like any proteins, are composed by 20 different amino acids, each of which is determined by specific triplet codon in their genes. Site-directed mutagenesis is a widely used molecular biology method to change the triplet in the coding sequence and thereby the amino acid residue in the protein sequence. Functional characterization of the ion channels carrying point mutations allows us to interrogate the stru...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Generation of Antibodies That Are Externally Acting Isoform-Specific Inhibitors of Ion Channels
There is demand for isoform-specific ion channel inhibitors as tools to investigate the biology of endogenous ion channels and validate them as targets in drug discovery programs. There is also hope that such inhibitors may be new therapeutic agents or provide the foundation for such agents. However, in practice, it is commonly experienced that inhibitors lack sufficient specificity, fail to distinguish between members of a class of ion channel, or have other (non-ion channel) off-target effects. Due to their extraordinary specificity, antibodies offer a potentially attractive strategy for overcoming these problems. I...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
Imaging and Quantification of Recycled KATP Channels
This chapter describes immunochemistry-based methods to investigate recycling of membrane proteins at the cell surface. Two methods are described, one qualitative and the other quantitative. Both methods consist of two rounds of extracellular antibody capture. Firstly, a primary antibody is captured by an extracellular epitope presented by the target membrane protein and is subsequently internalized. Secondly, the primary antibody-labelled protein is recycled back to the membrane where it is captured by a probe-conjugated secondary antibody. In the qualitative assay, the probe is a fluorophore, which can be imaged by ...
Source: Springer protocols feed by Cell Biology - April 12, 2013 Category: Cytology Source Type: news
