HAT medium

Grown In presence of HAT medium Hybridoma multiplies myeloma and B cells die... 

The cell fusion mixture is transferred to a culture medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Unflised myeloma cells are unable to grow as they lack HGPRT. Unflised normal spleen cells can grow but their proliferahon is limited and they eventually die out. The hybridoma cell can proliferate in the HAT medium as the normal spleen cell supplies the enzyme which enables the hybridoma to utilize extracellular hypoxanthine. 

Successful fusion (2) is a rare event, but the frequency can be improved by adding polyethylene glycol (PEG). To obtain only successfully fused cells, incubation is required for an extended period in a primary culture with HAT medium (3), which contains hypoxan-thine, aminopterin, and thymidine. Amino-pterin, an analogue of dihydrofolic acid, competitively inhibits dihydrofolate reductase and thus inhibits the synthesis of dTMP (see p. 402). As dTMP is essential for DNA synthesis, myeloma cells cannot survive in the presence of aminopterin. Although spleen cells are able to circumvent the inhibitory effect of aminopterin by using hypoxanthine and thymidine, they have a limited lifespan and die. Only hybridomas survive culture in HAT medium, because they possess both the immortality of the myeloma cells and the spleen cells metabolic side pathway. 

Feeder cells for fusion cultures (see Note 6) essential for fusions employing rat myelomas. Quickly thaw irradiated rat fibroblasts, prepared as described in Note 6, just before commencing the cell fusion. Add the cells to 10 mL of serum-free DMEM, centrifuge, and wash once in serum-free DMEM Resuspend feeders in HAT medium just before addition of the fusion mixture. Alternatively, use thymocytes from spleen donors (mouse). 

Discard PEG/RPMI mixture and replace with 10 mL of HAT medium. Do not disturb the cell pellet, then incubate the cell pellet for 5-10 min at 37°C. 

After 7 d examine the wells for the presence of hybridomas and given an additional 0.1 mL of HAT medium. 

HGPRT and TK enzymes are important in the synthesis of DNA from preformed nucleotides provided in the culture medium. The myeloma cells lacking these enzymes are unable to utilize the hypoxanthine or thymidine from the medium moreover, they die in the presence of aminopterin, which inhibits the de novo synthesis of DNA. Thus, in a medium containing aminopterin, hypoxanthine, and thymidine (HAT medium) only those hybridomas that receive the HGPRT or TK enzyme from the normal parents (spleen B lymphocytes) will survive. 

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). 

Incubate in a C02 incubator at 37°C. The myeloma cells cannot grow in HAT medium and the spleen cells are non-dividing so only fused cells will grow. 

Although neither TK. cells nor HPRT- cells will grow separately in HAT medium it is found that mixed populations of these two mutant lines will grow in HAT medium (see Fig. 13.3). 

Fig. 13.3. Metabolic cooperation between BHK21/C3 cells. Growth of BHK-HPRT-and BHK-TK- cells separately and in mixed (1 1) culture, in medium containing hypoxanthine, aminopterin and thymidine (HAT medium). HPRT = hypoxanthine phosphoribosyl transferase TK = thymidine kinase. (Reproduced from Pitts, 1971, with kind permission of the author and publisher.)...

HAT medium medium supplemented with hypoxanthine, aminop-terin and thymidine... 

HAT medium. Mutant cells unable to synthesize nucleotides by salvage pathways are very useful tools in molecular and cell biology. Suppose that cell A lacks thymidine kinase, the enzyme catalyzing the phosphorylation of thymidine to thymidylate, and that cell B lacks hypoxanthine-guanine phosphoribosyl transferase. 

Radiation propagates in the form of electromagnetic waves. The frequency e and wavelength A of elecirocnagnelic waves in a medium are telaterl by A = c/v. where c is the speed of propagation in (hat medium. All matter continuously emits thermal radiation as a result of vibrational and rotational niotioiis of molecules, atoms, and electrons of a substance. 

Cells are cultured in RPMI1640 with glutamine and 10% FBS. The cells are resistant to 10 mM 8-azaguanine and die in the presence of HAT medium. 

Figure 6-14 General method for > preparation of monoclonal antibod.e. using hybridomas and HAT medium Ali antibody Ag, antigen.

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