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Fluorescence-Activated Cell Sorting According to Receptor Density: Application for Isolating Transfected Cell Lines

Isolation of cells expressing transfected cDNAcan be extremely difficult and tedious if the efficiency of expression is low (less than 1–5%). If the receptor being selected for is a cell surface or integral membrane protein at least partly exposed to the exterior, then it is possible to use the extr ...

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Expression of Foreign Genes in Cultured Insect Cells Using a Recombinant Baculovirus Vector

An important consideration for the expression of cloned genes in recombinant expression systems is the ability of the foreign host to produce the protein faithfully in a form that is similar or identical to that found in the cell type from which the gene was cloned. For eukaryotic proteins, this freq ...

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Chloramphenicol Acetyltransferase as a Reporter in Mammalian Gene Transfer

Reporter genes can be used to advantage in a variety of different kinds of gene transfer experiments. One of the most common applications is the optimization of transfection methods. Owing to the large number of variables that influence the uptake and expression of exogenously introduced ge ...

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Immunizing Schedules for Hybridoma Production

Immunization protocols for generating activated B-lymphocytes suitable for hybridoma production vary widely. The optimal amount of antigen given, the way of antigen presentation, and the timing between injections must be determined for each antigen. Often, the antigen of interest ...

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Fusion Protocol for the Production of Mouse Hybridomas

Hybridoma technology makes it possible to produce almost unlimited amounts of monospecific antibodies. Each hybridoma represents only one of several B-lymphocytes responding to a particular antigen, and for this reason, monoclonal antibodies can also be produced against impure ...

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Cloning of Hybridomas

Hybrid clones will appear within 2–3 wk after fusion (see Chapter 46). It is of utmost importance that a newly established hybridoma is cloned thoroughly to ensure that the cells growing in the tissue culture are of monoclonal origin and not a mixture of two or more hybridomas. A mixture of cells may result in a ...

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Separation and Maintenance of Primary T and B Lymphocytes

Two distinct populations of lymphocytes have been identified: T lymphocytes, which are thymus-dependent, and B cells, first observed in the Bursa Fabricus of birds. Mammals do not have an equivalent structure, and there are varying opinions as to the similarity of these cells between species. ...

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Cryopreservation of Hybridomas

Hybridomas are exposed to many threats, such as contamination with bacteria and fungi, loss of chromosomes coding for antibody production, overgrowth by nonsecreting mutants, and cell death resulting from overgrowth. Therefore, newly established hybridomas should be frozen and s ...

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Enzyme-Linked Immunosorbent Assay for Screening of Antibodies in Hybridoma Supernatants

Enzyme-linked immunosorbent assay (ELISA) is a rapid and convenient method for screening of antibody producing hybridomas (1,2). The method is highly sensitive and can be applied to detect antibodies directed against soluble antigens as well as cell-bound antigens. Over a thousand cul ...

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Isotype Determination of Monoclonal Antibodies by Immunodiffusion

Immunodiffusion is an important qualitative method for the detection of antigens and antibodies. The technique may be used to determine the immunoglobulin heavy and light chain class and subclass of hybridoma antibodies. The monoclonal antibodies are allowed to diffuse toward anti ...

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Isoelectric Focusing and Immunofixation of Monoclonal Antibodies

Isoelectric focusing of monoclonal antibodies in agarose gel is one of several ways to characterize a hybridoma. Isoelectric focusing is performed on each monoclonal antibody, and the mouse immunoglobulin bands are immobilized by soaking the gel with rabbit antimouse immunoglobu ...

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Purification of Murine Monoclonal Antibodies

Hybridoma technology has made possible the production of highly specific, homogeneous antibodies with predefined binding characteristics, which can be produced in large amounts, from immortal cell lines. They probably represent the immunochemist’s ideal as reagents, and mono ...

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Large-Scale Production and Storage of Monoclonal Antibodies and Hybridomas

The end product of all the selection and cloning procedures described in Chapters 45–48 will be a monoclonal hybridoma culture growing in a 0.2-mL culture well. From this single well, sufficient cells must be grown for storage in liquid nitrogen. It is advisable that at least six ampules, divided bet ...

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Rat x Rat Hybridomas

There is an increasing interest in the preparation of rat � rat hybridomas, because they have been found to be more stable in culture than mouse hybridomas and they secrete consistently high levels (10 �g/mL and above) of monoclonal antibody. In addition, certain subclasses of rat IgG have been found ...

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Monoclonal Antibodies Against Glycosphingolipids (GSLs)-Gangliosides

Free GSL molecules are poorly immunogenic using conventional immunization procedures (1,2). Although immunogenicity has been increased by immunizing with purified glycolipids coated onto the surface of Salmonella minnesota organisms (3,4), this has not always been successf ...

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Mycoplasma Detection

Mycoplasma is the generic term used by cell biologists to denote organisms belonging to the Order Mycoplasmatales, which can infect cell cultures. Of particular interest are those organisms that belong to the Families Mycoplasmataceae (Mycoplasma) and Acholeplasmataceae (Ach ...

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Scale-Up of Suspension and Anchorage-Dependent Animal Cells

In this chapter, scale-up is described in a laboratory context (10–20 L), but the principles and techniques employed have been successfully adapted so that cells are now grown industrially in unit volumes of up to 8000 L for vaccine, interferon, and monoclonal antibody production. The need to scal ...

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Establishment of Lymphoblastoid Cell Lines

The ability to establish long-term B lymphocyte cultures from patients carrying particular genetic characteristics or with the ability to secrete specific antibodies (1) is an extremely valuable technique. However, there are several basic principles to follow in the approach to this ...

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Short-Term Chorionic Villi and Amniotic Fluid Cultures

Prenatal diagnosis of genetic disorders associated with specific biochemical, chromosomal, or molecular characteristics can be achieved from amniotie fluid (AF) or placenta (chorionic villus: CV) samples. Chorion material is usually obtained by sampling the placenta at the imp ...

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CFU-S: An Assay for Pluripotent Myelopoietic Stem Cells

The functional cells of the blood are short-lived; they are replaced continuously by proliferation and differentiation of hematopoietic precursors. Since cell division is required, exposure to agents that destroy proliferative potential is followed by loss or reduction in blood ...

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