Springer protocols feed by Immunology 
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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.Cytokine Detection by Flow Cytometry
Analysis of intracellular cytokines is extremely important in the clinical treatment of numerous diseases. Flow cytometry (FCM) is a highly effective technique that detects intracellular cytokines using specific fluorescence-labeled antibodies. The common steps of this assay include cell collection, fixation, permeabilization, blocking, intracellular staining and analysis by FCM. This technique also allows for analyzing the biological function of cytokines. In this chapter, we describe a modified method to detect the specific intracellular cytokine staining using FCM, with an emphasis on the effects of variables including ...
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
Intracellular Staining and Detection of Cytokines by Fluorescence-Activated Flow Cytometry
The detection of cytokines inside cells producing them has made a tremendous impact on the way immune reactivity is measured. Intracellular cytokine staining is the only immunological technique allowing determination of antigen-specific T cell function and phenotype at the same time; for this reason, it is one of the most popular methods to measure antigenicity in the evaluation of vaccine efficacy and in the study of infectious diseases. It is a flow cytometric technique based on staining of intracellular cytokines and cell markers (surface or cytoplasmic) with fluorescent antibodies after short term culture of stimulated...
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
Cytokine-Induced Neutrophil Chemotaxis Assay
Chemotaxis is directed migration of a cell type to a distant chemoattractant. When this chemoattractant is a cytokine, the term chemokine is often used. Chemotaxis by neutrophils, specifically polymorphonuclear leukocytes (PMNs), plays a critical role in the innate immune response. On an equimolar basis, interleukin-8 (IL-8) is one of the most potent PMN chemokines known. This chapter describes an in vitro chemotaxis technique using PMNs and IL-8 which can serve the investigator as an established model from which new studies can be developed. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
Control of Pro-inflammatory Cytokine Release from Human Monocytes with the Use of an Interleukin-1 Monoclonal Antibody
The monocytes (MONOs) can be considered as “double-edge swords”; they have both important pro-inflammatory and anti-inflammatory functions manifested in part by cytokine production and release. Although MONOs are circulating cells, they are the major precursors of a variety of tissue-specific immune cells such as the alveolar macrophage, dendritic cells, microglial cells, and Kupffer cells. Unlike the polymorphonuclear leukocyte, which produces no or very little interleukin-10 (IL-10), the monocyte can produce this potent anti-inflammatory cytokine to control inflammation. IL-10, on an equimolar basis, is a mor...
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
Use of shRNA for Stable Suppression of Chemokine Receptor Expression and Function in Human Cancer Cell Lines
In this chapter, we describe a protocol used for stable silencing of chemokine receptor CXCR7 in human cancer cells using shRNA in a lipid transfection setting, previously published by our laboratory. We provide thorough detail and background information about the process of shRNA to clarify the importance of this process. We use CXCR7 shRNA and scrambled sequence shRNA constructs cloned into a pRS plasmid under the control of a U6 promoter for stable expression. Human cancer cells are transfected with shRNA-pRS using Lipofectamine 2000. Cells stably expressing the shRNA are selected from transfected cultures following 2 w...
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
Detecting Tie2, an Endothelial Growth Factor Receptor, by Using Immunohistochemistry in Mouse Lungs
Immunohistochemical (IHC) staining is an invaluable, sensitive, and effective method to detect the presence and localization of proteins in the cellular compartment in tissues. The basic concept of IHC is detecting the antigen in tissues by means of specific antibody binding, which is then demonstrated with a colored histochemical reaction that can be observed under a light microscope. The most challenging aspect of IHC techniques is optimizing the precise experimental conditions that are required to get a specific and a strong signal. The critical steps of IHC are specimen acquisition, fixation, permeabilization, detectio...
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
Detection of CXCR2 Cytokine Receptor Surface Expression Using Immunofluorescence
The interleukin-8 (IL-8, CXCL8) chemokine, also known as the neutrophil chemotactic factor, is a cytokine that plays a key role in inflammatory response, cell proliferation, migration, and survival. IL-8 expression is increased not only in inflammatory disorders, but also in many types of cancer, including prostate cancer. IL-8 acts as a ligand for the C-X-C chemokine receptor 2 (CXCR2) protein present on the cell plasma membrane. Binding of the IL-8 ligand to the CXCR2 receptor results in an intracellular signaling pathway mediated by GTP binding proteins coupled to the receptor itself. Knowledge of the CXCR2 expression l...
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
Analysis of the Cell Surface Expression of Cytokine Receptors Using the Surface Protein Biotinylation Method
Cytokines are pleiotropic, low-molecular-weight proteins that regulate the immune responses to infection and inflammation. They stimulate the immune responses by binding to cytokine receptors on the cell plasma membrane. Thus, knowledge of the expression level of particular cytokine receptors on cell surface is crucial for understanding the cytokine function and regulation. One of the techniques to explore the membrane embedded cytokine receptors is cell surface biotinylation. Biotinylated surface proteins can be rapidly purified through the strong interaction between biotin and streptavidin. Here, we describe the procedur...
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
A Mix-and-Measure Assay for Determining the Activation Status of Endogenous Cdc42 in Cytokine-stimulated Macrophage Cell Lysates
Cytokine stimulations of leukocytes many times result in transient activation of the p21 Rho family of small GTPases. The role of these molecules during cell migration and chemotaxis is well established. The traditional approach to study the activation dynamics of these proteins involves affinity pull-downs that are often cumbersome and prone to errors. Here, we describe a reagent and a method of simple “mix-and-measure” approach useful for determining the activation status of endogenous Cdc42 GTPase from cell lysates. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
Evaluation of the Adverse Effect of Low Concentration of Cadmium on Interleukin-4 Induced Class Switch Recombination in Burkett’s Lymphoma Raji Cell Line
Affinity maturation of B lymphocytes, a process that includes somatic hypermutation and class switch recombination, initiates global DNA rearrangements. The interruption of this process has an adverse effect on human health and results in immunodeficiency and autoimmune disease. Class switch recombination is a fundamental factor of the human adaptive immunity. Evaluation of the class switch recombination efficiency is an important component of laboratory diagnostic of immunotoxic components. Here, we describe a method for testing the efficiency of the class switch recombination. Cultivation of Raji Burkett’s lymphoma...
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
Assessment of Cytokine-Modulated Proteasome Activity
This chapter presents two methods for assessment of proteasome function. The first is a modification of the standard fluorogenic peptide cleavage assay which takes into account the effect of ATP on proteasome activity. This method is described in both its macro and high throughput micro-assay forms. The second is the Proteasome Constitutive Immuno-Subunit (active site) ELISA or ProCISE method. ProCISE is a modification of active site directed probe analysis and allows for convenient differentiation between active constitutive and immuno-subunits. While the utility of measuring proteasome activity and its relationship to cy...
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
Assessment of Phagocytic Activity of Cultured Macrophages Using Fluorescence Microscopy and Flow Cytometry
Phagocytosis is the process by which phagocytes, including macrophages, neutrophils and monocytes, engulf and kill invading pathogens, remove foreign particles, and clear cell debris. Phagocytes and their ability to phagocytose are an important part of the innate immune system and are critical for homeostasis of the host. Impairment in phagocytosis has been associated with numerous diseases and disorders. Different cytokines have been shown to affect the phagocytic process. Cytokines including TNFα, IL-1β, GM-CSF, and TGF-β1 were found to promote phagocytosis, whereas high mobility group box-1 (HMGB1) inhib...
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
Chemotactic Responses by Macrophages to a Directional Source of a Cytokine Delivered by a Micropipette
Macrophages, which are organized throughout every tissue, represent a key component of the immune system and the recruitment of macrophages to specific sites is important in normal host defense. However, when inappropriately recruited macrophages may damage or destroy healthy tissue; this is seen in several autoimmune diseases such as arthritis. Many cytokines, including CSF-1 and chemokines, are often upregulated in inflamed tissues and can induce the directional migration of macrophages towards the highest concentration of the cytokine in a process called chemotaxis. Chemokines were first described as chemoattractant cyt...
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
An In Vitro One-Dimensional Assay to Study Growth Factor-Regulated Tumor Cell–Macrophage Interaction
We describe an in vitro motility assay that combines time-lapse wide-field microscopy and micro-patterned linear adhesive substrates to reconstitute the in vivo behavior between macrophages, tumor cells, and ECM fibers in orthotopic rodent tumor models observed by intravital imaging. Commercially available linear stripes of 650 nm dye-labeled fibronectin microlithographed onto glass cover slips are sequentially plated with fluorescently labeled MTLn3 tumor cells and bone marrow-derived macrophages and time-lapse imaged for up to 8 h. Incubation with pharmacological inhibitors during the assay can identify important paracri...
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
A Useful Guide for Analysis of Immune Markers by Fluorochrome (Luminex) Technique
The fluorochrome Luminex technique is a bead-based sandwich immunoassay that combines the enzyme-linked immuno sorbent assay (ELISA) with flow cytometry. The Luminex technique allows multiple cytokines to be measured simultaneously in small volumes and provides a convenient and sensitive tool for the detection of a large number of, e.g., extracellular secreted cytokines to characterize cytokine profiles. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
