Bidstrupday7393

Staining cells or tissues with basic dyes was the mainstay of mast cell and basophil detection methods for more than a century following the first identification of these cell types using such methods. These techniques have now been largely supplanted by immunohistochemical procedures with monoclonal antibodies directed against unique constituents of these cell types. Immunohistochemistry with antibodies specific for the granule protease tryptase provides a more sensitive and discriminating means for detecting mast cells than using the classical histochemical procedures, and using antibodies specific for products of basophils (2D7 antigen and basogranulin) has allowed detection of basophils that infiltrate into tissues. The application of immunohistochemistry to detect more than one marker in the same cell has underpinned concepts of mast cell heterogeneity based on differential expression of chymase and other proteases. The double labeling procedures employed have also provided a means for investigating the expression of cytokines and a range of other products. Protocols are here set out that have been used for immunohistochemical detection of mast cells and basophils and their subpopulations in human tissues. Consideration is given to pitfalls to avoid and to a range of alternative approaches.Basophils and mast cells are known for their capability to release both preformed and newly synthesized inflammatory mediators. In this chapter, we describe how to stimulate and detect histamine released from basophils in whole blood, purified basophils, in vitro cultured mast cells, and in situ skin mast cells (the latter by microdialysis), using either a solid phase assay or flow cytometry. We also give an example of an activation protocol for basophil and mast cell cytokine release and discuss approaches for cytokine detection.Basophils have been suggested to express low quantities of RNA, challenging the study of gene expression within these cells. However, the purification technique employed might have an impact on the quantity and quality of RNA purified from basophils. This chapter describes a method which gives an optimal RNA output using a TRIzol-based method in contrast to a commercial kit.The mast cell (MC) activation assay is a robust in vitro tool for exploring MC reactivity in allergy. Here we describe the use of the mast cell activation test (MAT) that makes use of human primary MCs generated from peripheral blood progenitors, sensitized overnight with patients' sera and activated with allergens. Flow cytometry is used to assess the changes in expression of the activation marker CD63, and the percentage of cell degranulation is defined as the percentage of CD63+-positive MCs.Basophils and mast cells (MCs) are important effector cells in the immune system. For a long time, it has been known that these cells can be activated though the cross-linking of IgE antibodies bound to their high-affinity receptor (FcεRI). However, evidence has accumulated suggesting that these cells can also be activated by various IgE-independent mechanisms. Occupation of MAS Related GPR Family Member X2 (MRGPRX2), a G protein-coupled receptor, is described as an alternative IgE-independent activation mechanism. Here we describe a flow cytometric technique to analyze MRGPRX2 expression and its functionality on cultured human MCs and conditioned basophils, that is, basophils with upregulated surface expression of MRGPRX2.The basis of traditional flow cytometry allergy diagnosis is measurement of the expression of basophilic surface activation and/or degranulation markers. Basophils, upon encounter with a specific allergen that cross-links surface FcRI-bound IgE antibodies, not only secrete and release quantifiable bioactive mediators but also upregulate the expression of different markers (e.g., CD63, CD203c) which can be detected by multicolor flow cytometry using specific monoclonal antibodies. Here, we describe a novel technique that relies upon the staining of exteriorized anionic proteoglycans from a basophil granule matrix by cationic fluorescent avidin probes.

Correct diagnosis of immediate drug hypersensitivity reactions (IDHRs) can pose a significant challenge, mainly because of the absence of reliable in vitro tests, uncertainties associated with skin testing, and incomplete understanding of the underlying mechanisms.

To summarize and hypothesize on the potential of basophil activation test (BAT) as a safe aid to explore the mechanistic endotypes of IDHR, to identify antibody recognition sites, and to monitor drug desensitization.

A literature search was conducted using the keywords "allergy, basophil activation, CD63, CD203c, diagnosis, drugs, hypersensitivity, flow cytometry"; this was complemented by the authors' own expertise.

At present BAT has mainly been employed as a diagnostic aid. However, evidence is emerging that the technique might also deepen our insights in immune (allergic) and nonimmune (nonallergic) mechanistic processes of IDHR. It is anticipated that BAT might also benefit the identification of antibody recognition sites and benefit our understandings of desensitization strategies.

Although the nondiagnostic application of BAT in IDHR is still in its infancy, with increasing employment, we can expect the technique to become a valuable asset to study many domains of IDHR that remain poorly understood.

Although the nondiagnostic application of BAT in IDHR is still in its infancy, with increasing employment, we can expect the technique to become a valuable asset to study many domains of IDHR that remain poorly understood.The basis of flow cytometric allergy diagnosis is the quantification of changes in the expression of basophilic surface membrane markers (Ebo et al., Clin Exp Allergy 34 332-339, 2004). LY2603618 chemical structure Upon encountering specific allergens recognized by surface receptor FcεRI-bound IgE, basophils not only secrete and generate quantifiable bioactive mediators but also upregulate the expression of different markers (e.g., CD63, CD203c) which can be detected by multicolor flow cytometry using specific monoclonal antibodies (Ebo et al., Cytometry B Clin Cytom 74 201-210, 2008). Here, we describe two flow cytometry-based protocols which allow the detection of surface marker activation (Method 1) and changes in intragranular histamine (Method 2), both reflecting different facets of basophil activation.