Human mast cells (HMCs) are derived from a CD34+ pluripotent progenitor cell that is Kit (CD117+), CD13+, FcεRI- and lacks lineage-specific surface markers. Bone marrow and peripheral blood are two tissue sources available for obtaining CD34+ progenitor cells from which to culture HMCs. CD34+ ...

The survival of hemopoietic stem cells in culture is suppressed by various cytokines and stimuli. The development of human mast cells also is affected by these stem cell-inhibitory mechanisms because it requires a much-longer period as compared with the development of other cell lineages. ...

Nuclear factor-κB (NF-κB) is a key transcription factor that regulates the expression of genes involved in immune and inflammatory responses and in cell death and survival. This chapter describes in detail the method for measuring the NF-κB binding activity in the cultured human mast cell li ...

The biological functions of mast cells are regulated by several protein kinases, including the tyrosine kinases Fyn, Lyn, Syk, and FAK and the serine/threonine kinases Akt and PKC α/β. The mitogen-activated protein kinases extracellular signal-regulated kinases, JNK, and p38MAPK also ...

Human mast cells are capable of secreting a plethora of inflammatory mediators and cytokines that may play a pivotal role in innate immune and inflammatory responses. Activation of mast cells by antigen and immunoglobulin E (IgE) results in signaling, gene expression, and expression of inf ...

Mast cells are the crucial effector cells for allergic reactions. They are activated through the aggregation of the high-affinity immunoglobulin E (IgE) receptor (FcεRI) with allergen and allergen-specific IgE. Tyrosine phosphorylation of FcεRI subunits and various signaling p ...

Human mast cells contain proteases that are important functional components and serve as markers of mast cell activation or degranulation. Although tryptase is the best recognized mast cell protease, chymase and Cathepsin G also are found in some human mast cells. Methods for measuring the ...

Mast cells are crucial to the development of chronic allergic inflammation and are likely to play a critical role in host defense. In this chapter methodology for histamine and cytokine assays is provided. Crosslinkage of IgE receptor I (FcεRI) on cord bloodderived mast cells by myeloma IgE and a ...

Mast cells are highly responsive cells that are capable of secreting a variety of inflammatory mediators, including histamine, heparin, serine proteases, leukotrienes, prostaglandins, and thromboxanes. Studies from several laboratories have demonstrated that mast cells h ...

When using a model to study disease, it may be advantageous to identify molecules responsible for biologic functions observed in the model to better understand the disease process being studied. The late phase reaction is used as a model for chronic inflammation, and the histamine releasing a ...

The purpose of this chapter is to review the analytical methodology specifically associated with studying the role of nitric oxide (NO) in mast cell physiology and biochemistry. The methodology for measuring cellular secretion of nitric oxide with Griess Reagent will be described in det ...

The relative rarity of mast cells (MCs) and the rich content of heparin in the cytoplasmic granules of MCs pose technical challenges in reliably detecting growth factors (GFs) or cytokines in MCs by conventional immunohistological stain (IHS) methods. A variety of polypeptide growth fact ...

Experimental autoimmune encephalomyelitis (EAE) and Theiler’s murine encephalitis virus-induced demyelinating disease (TMEV-IDD) are two clinically relevant murine models of multiple sclerosis (MS). Like MS, both are characterized by mononuclear cell infiltrate into t ...

Various methods have been devised to detect antibody-producing cells in culture and also determine the isotype of the immunoglobulin produced. We have found the ELISPOT assay to be the method of choice for detecting human monoclonal antibodies (MAbs) produced by heterohybridomas (see ...

One of the easiest and most sensitive methods to screen for, to quantify, and to determine the quality of antibodies in the serum of mice, the spleens of which may be used for fusions, is by solid-phase enzyme-linked immunoassays (EIA) (1,2). In these procedures, the wells of 96-well plates or strip plates are ...

Immunohistochemistry (IHC) or immunocytochemistry is a method for the localization of specific antigens in tissues or cells using antibody probes. Because it provides detailed and specific information on the expression of cell markers, the IHC technique has wide application in the i ...

Immunoelectron microscopy (IEM) techniques, usually in conjunction with light microscopy techniques, are used for the localization of antigenic molecules that may be employed for vaccine development, organelle characterization, or other biologically relevant applica ...

Protein blotting involves the immobilization of proteins on a support medium. The most usual format is the electrophoretic transfer of proteins resolved on a sodium dodecyl sulfate (SDS)-polyacrylamide gel to a membrane, which is known as Western blotting (1). Polyacrylamide gel elect ...

Recent advances in the design of flow cytometers and the development of associated software have facilitated the use of flow cytometry as a primary method for identifying and characterizing monoclonal antibodies (MAbs) reactive with molecules expressed on one or more lineages of leuk ...

Mammalian cells from every tissue have many different types of proteins on their extracellular membranes that maintain their functional integrity. These molecules participate in communication, in metabolism, in locomotion, or in adherence. Antibodies developed against the ...