HAT selection medium

Centrifuge for 3 min at 400g, and then resuspend the cells in 200 mL HAT selection medium, and add irradiated fibroblast or thymocyte feeder cells. Plate 2-mL aliquots into four 24-well plates or, if necessary, five 96-well plates (fusions with SP2/0 myeloma) (see Note 6). 

The ability of cells to uptake thymidine and phosphorylate it is exploited in the so-called HAT selection medium (see here), which selects for cells that have functional nucleotide salvage pathways. 

A number of protocols have been described for the generation of mouse hybrid-omas and workers have their own particular protocol. We have used a standard procedure for the fusion of mouse and rat myelomas with considerable success over the last twenty years and this is described in Protocol 7. Basically, 10 lymphocytes are mixed with 2 x 10 mouse myeloma cells or 5 X 10 Y3 cells in a round-bottomed tube and pelleted by centrifugation. Then the ceUs are fused by the addition of 1 ml of 50% PEG 1500 and plated into HAT selection medium to allow the growth of the hybridomas generated. 

Centrifuge for 3 min at 400 g, then resuspend the cells in 200 ml of HAT selection medium (containing feeder cells where necessary), and plate 2 ml aliquots into four 24-well plates or 200 pi aliquots into ten 96-well plates. Incubate at 37 °C in 5% C02-... 

Figure 6-38 depicts the general procedure for generating and selecting hybridomas. In this case, normal B lymphocytes are fused with myeloma cells that cannot grow In HAT medium, the most common selection medium used In the production of hybridomas. Only the myeloma-lymphocyte hybrids can survive and grow for an extended period In HAT medium for reasons described shortly. Thus, this selection medium permits the separation of hybridoma cells from both types of parental cells and any... 

HAT Selection - The compounds hypoxanthine, aminopterin (see here), and thymidine (H,A, and T, respectively) can be used to select for cells having functional salvage pathways. Aminopterin inhibits dihydrofolate reductase, which blocks de novo purine and thymidine synthesis. Only cells which can utilize thymidine (pyrimidine salvage) and hypoxanthine (purine salvage) can grow in this medium. 

FIGURE 52-2 Generation of monoclonal antibodies. Mice are immunized with the selected antigen, and spleen or lymph node is harvested and B cells separated. These B cells are fused to a suitable B-cell myeloma that has been selected for its inability to grow in medium supplemented with hypoxanthine, aminopterin, and thymidine (HAT). Only myelomas that fuse with B cells can survive in HAT-supplemented medium. The hybridomas expand in culture. Those of interest based upon a specific screening technique are then selected and cloned by limiting dilution. Monoclonal antibodies can be used directly as supernatants or ascites fluid experimentally but are purified for clinical use. HPRT, hypoxanthine-guanine phosphoribosyl transferase. 

Figure 3 Schematic representation of HAT selection. HAT medium contains aminopterin to block the de novo pathway and hypoxanthine and thymidine to allow growth by the salvage pathway. Cells that lack either HGPRT or TK will die in HAT medium, because they lack the ability to use the salvage pathway to synthesize nucleic acids.

The selective medium, HAT contains hypoxanthine, aminopterin, and thymidine. This medium allows the selection and growth of hybridomas which are HGPRT+ and have a normal functioning salvage pathway. However, HAT is unable to support the growth of the HGPRT" myelomas because the de novo pathway is inhibited and the salvage pathway cannot function because of the defective enzyme. 

A portion of the cells dispersed from these same resistant colonies was plated into nonselective culture medium and allowed to grow for approximately 10 generations. Cells were then trypsinizcd and plated at cloning densities into three different types of media (1) HAT medium, i.e., Ham s FIO that had been prepared without HX and thymidine but was now supplemented with 30 mM HX (H), 0.1 aminopterin (A), and 30 nM thymidine (T) and 10% FCS (2) nonselective culture medium, i.e., FIO lacking HX but supplemented with 10% FCS and (3) the AG or TG selective medium described in Section 2.1. Growth in HAT medium requires a cell to possess HPRT activity (or, alternatively, to be resistant to aminopterin). Growth in AG or TG selective medium requires a cell to lack HPRT activity or to have altered uptake of these purines. After 3 weeks, the dishes were stained and the number of clones in each set determined. 

Clearly this places severe limitations on the use of HAT as a selective medium against HGPRT cells. We have not yet ascertained whether these HGPRT mutants will show the adaptation to aminopterin described by Riccardi and Littlefield (23). 

Select HPGRT+ cells by culturmg the mixture in a medium containing HAT (hypoxanthine, aminopterin, and thymidine), which is growth inhibitory to HPGRT+ cells. Therefore, the myeloma cells will die in this medium while the hybridoma cells proliferate. The unfused lymphocytes will die due to their limited lifespan. 

Human cells are killed by 10 7 ouabain, but mouse cells are relatively resistant in that 10 3M is a lethal dose. Mouse human hybrids show intermediate sensitivity and hence HAT medium containing 10 7M ouabain can be used to select for hybrids between any human cell and TK or HPRT mouse cells. This enables primarily human cells and other non-selected strains to be used in fusion experiments (Mayhew, 1972 Thompson and Baker, 1973). 

Other selection systems involve the fusion of proline requiring CHO cells and defective mouse cells in HAT medium lacking proline and the fusion of normal lymphocytes (which do not grow in vitro) with defective mouse cells (which do not grow in HAT medium). It is this latter technique which has allowed the isolation of clones of cells which will synthesise in vitro large amounts of a single antibody (i.e. a monoclonal antibody) (Kohler, 1982). 

Hybridomas are generated 3 d following an iv booster injection of 10 pg of DT in phosphate-buffered saline (PBS) and selected using Hy medium with 10% fetal calf serum plus hypoxanthine, aminopterin, and thymidine (HAT) (6,16). 

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