HAT Medium (hypoxanthine-aminopterin-thymidine medium) is a selection medium for mammalian cell culture, which relies on the combination of aminopterin, a drug that acts as a powerful folate metabolism inhibitor by inhibiting dihydrofolate reductase, with hypoxanthine (a purine derivative) and thymidine (a deoxy
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What is the principle of HAT selection?
HAT selection depends on the fact that mammalian cells can synthesize nucleotides by two different pathways: the de novo and the salvage pathways. The de novo pathway in which a methyl or formyl group is transferred from an activated from of tetrahydrofolate, is blocked by Aminopterin, a folic acid analog.
What is the principle of HAT selection in hybridoma technology?
Hybridoma selection using HAT medium
During the fusion process, three types of cells are present: (1) unfused myeloma cells that are deficient in an enzyme called HGPRT, (2) unfused spleen cells, and (3) fused hybridoma cells. Unfused spleen cells are easily selected against since they do not replicate in culture.
What is the purpose of HAT medium in monoclonal antibody production?
The purpose of the medium is to: (1) selectively kill unfused myeloma cells that are well adapted to tissue culture and would otherwise outgrow any hybridomas produced and (2) eliminate any myeloma-myeloma hybridomas that lack HPRTase.
How do you make a hat media?
Hypoxanthine, aminopterin, and thymidine selection (HAT) medium is commonly prepared from two stock solutions, 100× HT and 100× A. 1. To prepare 100 mL of 100× HT, dissolve 136 mg of hypoxanthine and 38 mg of thymidine in 100 mL of H2O suitable for tissue culture.
What is the purpose of HAT medium?
HAT medium is used for preparation of monoclonal antibodies. This process is called hybridoma technology. Laboratory animals (e.g., mice) are first exposed to an antigen against which we are interested in isolating an antibody.
Who introduced HAT medium?
Littlefield
HAT medium originally developed by Littlefield in 1964 (1) has been one of the key factors that has made hybridoma generation practical. The value of this medium is generally considered from the viewpoint of its ability to inhibit unfused myeloma cell proliferation.
Why hybridoma cells survive in HAT medium?
Hybridoma cells: These cells survive in the medium as they have enzyme HGPRT inherited from spleen cells. Hence they successfully alternate to salvage pathway in case de novo synthesis is blocked by aminopterin.
What is the purpose of hybridoma?
Hybridoma is a culture of hybrid cells that results from the fusion of B cells and myeloma cells. Hybridoma technology produces hybridomas. This technology was developed to produce mAbs. Hybridomas possess two important properties of B cells, production of antibodies, and immortalization of myeloma cells.
Why does myeloma cells are not grown in HAT medium?
Unfused myeloma cells cannot grow because they lack HGPRT and thus cannot replicate their DNA.
What are the application of monoclonal antibodies?
Monoclonal antibodies can be used alone or to carry drugs and radioactive or toxic substances directly to cancer cells. Monoclonal antibodies that are used as drugs assist the natural immune system’s function in fighting cancer. These medications may be used in combination with other cancer treatments.
How is monoclonal antibody produced?
The traditional monoclonal antibody (mAb) production process usually starts with generation of mAb-producing cells (i.e. hybridomas) by fusing myeloma cells with desired antibody-producing splenocytes (e.g. B cells). These B cells are typically sourced from animals, usually mice.
Which of the following statement is correct regarding HAT medium?
Correct Option: B
Therefore, the use of HAT medium for cell culture is a form of artificial selection for cells containing working TK and HGPRT.
Who proposed hybridoma technology?
Georges Kohler and Cesar Milstein in 1975 invented the hybridoma technology, for which they received the Nobel Prize in 1984 in physiology and medicine [25].
What is the role of HGPRT in hybridoma technology?
The key concept is that the hybridoma receives a functional HGPRT gene from the primary B cell, thus enabling the survival of hybridomas vs. myeloma cells. While primary B cells can survive for a time in culture and produce antibody, they will eventually die without any additional selection.
How hybridoma cells are produced?
Hybridoma technology is one of the most common methods used to produce monoclonal antibodies. In this process, antibody-producing B lymphocytes are isolated from mice after immunizing the mice with specific antigen and are fused with immortal myeloma cell lines to form hybrid cells, called hybridoma cell lines.
WHAT IS A hybridoma cell?
Hybridomas are cells formed via fusion between a short-lived antibody-producing B cell and an immortal myeloma cell. Each hybridoma constitutively expresses a large amount of one specific mAb, and favored hybridoma cell lines can be cryopreserved for long-lasting mAb production.
WHAT IS A hybridoma cell line?
A hybridoma is a cell line arising from one hybrid cell that is capable of secreting a monoclonal antibody specific to one epitope of your antigen permanently in culture.
What are the limitations of hybridoma technology?
Another important limitation of hybridomas consists in the origin of the plasma cells. Initially produced from mouse cell lines, these antibodies were soon found to have limited therapeutic application due to their high immunogenicity.
What is the purpose of using myeloma cells?
Multiple myeloma cells are abnormal plasma cells (a type of white blood cell) that build up in the bone marrow and form tumors in many bones of the body. Normal plasma cells make antibodies to help the body fight infection and disease. As the number of multiple myeloma cells increases, more antibodies are made.
What are two uses of monoclonal antibodies?
Monoclonal antibodies are used for diagnosis, disease treatment and research. They’re used: As probes to identify materials in laboratories or for use in home-testing kits like those for pregnancy or ovulation. To type tissue and blood for use in transplants.