Tierneyvestergaard3758

The use of flow cytometry allows simultaneous measurement and multiparametric analysis of single cells in a heterogenous solution. The purpose of flow cytometry can vary depending on the use of antibodies and dyes targeted for specific cell molecules. The method of immune-phenotyping with fluorescently conjugated antibodies to label cell proteins or DNA works in tandem with fluidic, optic, and electrical systems present in the flow cytometer. Some flow cytometers can detect numerous fluorescent molecules on a single cell, allowing the measurement of more than 30 parameters. This ability to detect, measure, and quantitate multiple fluorescent markers on a single cell makes the flow cytometer a useful tool for analyzing various aspects of cell phenotype and function. Here we describe a standardized protocol for surface and intracellular immune-phenotyping of murine lungs, beginning with the building of an optimal antibody panel and ending with data analysis and representation, including sample gating strategies for innate and adaptive immune responses.Flow cytometry is a popular technique used for both clinical and research purposes. It involves laser-based technology to characterize cells based on size, shape, and complexity. Additionally, flow cytometers are equipped with the ability to take fluorescence measurements at multiple wavelengths. This capability makes the flow cytometer a practical resource in the utilization of fluorescently conjugated antibodies, fluorescent proteins, DNA binding dyes, viability dyes, and ion indicator dyes. As the technology advances, the number of parameters a flow cytometer can measure has increased tremendously, and now some has the capacity to analyze 30-50 or more parameters on a single cell. Here, we describe the basic principles involved in the mechanics and procedures of flow cytometry along with an insight into applications of flow cytometry techniques for biomedical and allergic disease research.Type-I hypersensitivity is commonly characterized by increased levels of antigen-specific immunoglobulin (Ig) E. Therefore, it is important for clinical and research investigators to reliably measure serum levels of IgE in allergic patients and animal models. While current ELISA-based methods are simple and commonly performed for the detection of allergen-specific IgE using serum or plasma, they may produce misleading results. This is in part due to decreased sensitivity for IgE in the presence of other Ig isotypes in the same sample, such as IgG, that are typically more abundant than IgE. When assessment of multiple Ig isotypes is necessary, performing optimized assays for individual isotypes requires high sample volumes. Here, we describe an approach to increase the sensitivity for IgE detection while conserving the sample volume needed. This method not only improves the accuracy of serum IgE measurements but also allows simultaneous analysis of other allergen-specific immunoglobulins.The regulation of vascular permeability is critical in inflammation. compound library chemical It controls the distribution of water and plasma contents such as immunoglobulins in peripheral tissues. To regulate allergic diseases, it is important to study vascular biology especially in inflammation. Since the vascular permeability changes in minutes upon the exposure to proinflammatory mediators, intravital imaging system is a powerful technique to capture such dynamic responses. We here describe how to evaluate vascular permeability in vivo using multiphoton microscopy. We use various sizes of fluorescence-labeled dextran to visualize how leaky the blood vessels are in the steady state and in inflammation. Using this assay system, we can illustrate the dynamic kinetics of vascular permeability in vivo in real-time. This assay system provides a novel convenient way to study vascular biology that is beneficial in the assessment of various animal models of allergic disease.Mouse models of allergic conjunctivitis mimic various aspects of human allergic conjunctivitis. They are useful as acute models of allergic conjunctivitis to study immunological aspects of this condition. In this chapter, we will describe ragweed-pollen-induced experimental allergic conjunctivitis (mostly driven by adaptive immunity), and papain-soaked contact lens-induced experimental allergic conjunctivitis (mostly driven by innate immunity). Giemsa staining of histological sections is used for quantification of the number of infiltrating eosinophils, which is useful to evaluate the severity of the allergic inflammation. Immunohistochemical staining and quantitative PCR are used to clarify spatiotemporal expression of proinflammatory molecules in the conjunctival tissue. Flow cytometric analysis of conjunctival tissue is used for the detection of innate lymphoid cell type 2 (ILC2) in the ocular surface tissues.IL-22 is an IL-10 family cytokine that is increased in asthma and atopic dermatitis (AD). However, the specific role of IL-22 in the pathogenesis of allergic lung inflammation and AD in vivo has yet to be elucidated. We aimed to develop mouse models of allergic diseases in the lung and skin with inducible and tissue-specific expression of IL-22, using a tetracycline (Tet)-controlled system. In this chapter, we describe a series of protocols we have developed to generate a construct that contains the TRE-Tight promoter and mouse IL-22 cDNA based on this system. Furthermore, we describe how to generate TRE-Tight-IL-22 mice through pronuclear microinjection. In our approach, two Tet-on (CC10-rtTA or SPC-rtTA) and a Tet-off (K5-tTA) transgenic mouse lines are selected to crossbreed with TRE-Tight-IL-22 mice to generate inducible tissue-specific transgenic lines. The transgenic strains, CC10-rtTA/TRE-Tight-IL-22 (CC10-rtTA-IL-22) or SPC-rtTA/TRE-Tight-IL-22 (SPC-rtTA-IL-22) mice, do not produce detectable levels onovel therapeutics in the fields of inflammation, asthma, and allergic dermatitis.Mouse models of allergic asthma have been utilized to establish the role of T helper type 2 (Th2) cells in driving lung inflammation, airway hyperresponsiveness, and obstruction. Here, we present the allergic asthma models, in which mice are hypersensitized to ovalbumin (OVA) and house dust mite (HDM). These models mimic the major characteristics of human asthma including the eosinophilic inflammation and hyperactivity of the airway, overproduction of Th2 cytokines in the lung, and elevated total and allergen-specific immunoglobulin E (IgE) in serum.