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Flow Cytometric Analysis of Cell Surface Antigen Density

Analysis of cell surface antigen density and, thus, the distribution of cell surface molecules among individual members of complex cell populations can be achieved rapidly and accurately by flow cytometry. This approach has been revolutionized over the last 20 yr by the codevelopment of m ...

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Multiple Immunofluorescence Analysis of Cells Using Flow Cytometry

As indicated in Chapter 33, flow cytometry has developed rapidly to provide a powerful means of characterizing complex cell populations, both in terms of quantitative analysis of functional cell-associated molecules, and, as is considered here in more detail, the simultaneous analys ...

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Flow Cytometric Methods of Analyzing Apoptotic Cells

Apoptosis is a physiological, programmed mode of cell death, which is necessary for tissue modeling and organogenesis in embryonic development and in the control of homeostasis in a diversity of tissue types (1). The distinct morphological features of apoptosis clearly distinguish it f ...

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Sorting of Human Peripheral Blood T-Cell Subsets Using Immunomagnetic Beads

Immunomagnetic beads are uniform, polymer particles coated with a polystyrene shell that provides both a smooth, hydrophobic surface to facilitate absorption of molecules, such as antibodies, and surface hydroxyl groups that allow covalent chemical binding of other bioreactive ...

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Flow Cytometric Detection of Proliferation-Associated Antigens, PCNA and Ki-67

The rapid evolution of monoclonal antibody (MAb) technology has resulted in an ever-growing repertoire of proteins becoming accessible for study using immunohistochemical techniques. Nuclear antigens represent a class of proteins of increasing significance and diversity; ...

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Preparation and Use of Nonradioactive Hybridization Probes

Molecular hybridization is a useful technique for identifying specific target sequences even when they are present as a single copy in a complex population of highly heterogeneous gene sequences. It can be performed either on a solid matrix on which pure DNA (or RNA) is bound (blot hybridizatio ...

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Cellular Human and Viral DNA Detection by Nonisotopic In Situ Hybridization

In situ hybridization may be defined as the detection of nucleic acids in situ in cells, tissues, chromosomes, and isolated cell organelles. The technique was described in 1969 by two separate groups who demonstrated repetitive ribosomal sequences in nuclei of Xenopus oocytes using radio ...

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Production and Characterization of Antibodies Against Synthetic Peptides

Immunization protocols vary greatly among laboratories. In general, there are no hard and fast rules, and most protocols give satisfactory results. The methods described below are designed to give optimal results with minimal injury to the test animal, and we have used them extensively and s ...

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Expression of Recombinant Antibody Fusion Proteins in E. coli

Conjugates of antibodies or antibody fragments with foreign proteins have attracted interest because of their therapeutic potential as antitumor agents in vivo. Localization of the complex to the target site is dependent on the specificity of the antibody moiety, and local cytotoxic a ...

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Nonisotopic In Situ Hybridization: Gene Mapping and Cytogenetics

With the advent of nonradioactive probes during the past decade, in situ hybridization has become an immensely valuable tool in the hands of: 1. Developmental biologists and cell biologists for the detection of mRNA in cells and tissue sections (1

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Antibody Screening of Bacteriophage gt-11 DNA Expression Libraries

Much of our current understanding of the molecular details of the activity and interactions of proteins has stemmed from the ability to isolate cDNA encoding these proteins. Computer-based analysis of deduced amino acid sequence allows predictions to be made about their structure and fu ...

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Expression of Antibody Fusion Proteins in Mammalian Cells

Recombinant DNA technology provides a means to mimic nature and build multidomain proteins that display several functions. This ability is being applied by the pharmaceutical industry to develop novel products. Fusion proteins of antibodies and enzymes formed one of the earliest exa ...

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Display of Antibody Chains on Filamentous Bacteriophage

The construction of very large combinatorial libraries whereby antigen-binding functional domains of antibodies (Abs) are cloned and displayed on the surface of bacteriophage is proving to be a powerful technology in providing reagents for immunotherapy. Combinations of rand ...

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Production of Human Fab Antibody Fragments from Phage Display Libraries

Phage display involves expression of a large library of diverse molecules as fusion proteins on the surface of filamentous bacteriophage. In this form, they may be subjected to biological or molecular selection to isolate molecules with the desired binding properties (1) (see review in ref. ...

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Modulation of the Interferon- Signal by Transfection of Cells with an Antisense-RNA Expressing Vector

Apart from cell-cell contacts, signal transduction mechanisms active during immune defense are mainly regulated by cytokines. They accomplish their pleiotropic, paracrine, and/or autocrine biological activities as a result of the contact with a membrane receptor followed by si ...

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Competitive RT-PCR to Quantify Small Amounts of mRNA

Monitoring cytokine responses to various stimuli or determining the expression pattern of cytokine mRNAs requires sensitive technologies for cytokine mRNA quantitation where there is a limited quantity of material, for example, when working with biopsies. Owing to their amplif ...

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Purification, Identification, and Synthesis of Chemokines

Chemokines, i.e., low-molecular-weight chemotactic cytokines, are generally produced in very small amounts. Hence, liters of conditioned medium from primary cells or cell lines are necessary as start material to obtain microgram amounts of natural chemokine (see Note 1). A substanti ...

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In SituHybridization for Cytokines in Human Tissue Biopsies

In situ hybridization (ISH) involves hybridization of labeled nucleic acid probes with the target mRNA. As cytokines are soluble mediators exported from the cells in which they are produced, ISH is a powerful technique that can be used to identify the exact identity and location of the cells that p ...

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Receptor Isolation and Characterization: From Protein to Gene

Affinity chromatography as it is known today, namely, the concept and the immense power of biorecognition as a means of purification, was introduced in 1968 by Cuatracasas, Wilchek, and Anfinsen (1). This technique is used in 60% of all purification protocols (2). Almost any given biomolecule that ...

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Crystallization of Cytokine-Receptor Complexes

Crystallography is the most powerful method for determining the three-dimensional structure of complicated biological molecules. As the technology has improved, larger and more complicated structures involving previously unobtainable proteins have become available. ...

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