Mouse hybridoma

Vitamins and lipids are often required for animal cells to grow in serum-free medium. Phosphoethanolamine and ethanolamine are key additives that facilitate the growth of the mammary tumor cell line 64024 (Kano-Sueoka and Errick, 1981). In addition, ethanolamine promotes the growth of human lymphocytes and mouse hybridoma cells. Short-term cultures of human diploid lung and foreskin fibroblasts grow in medium that includes among its supplements soybean lecithin, cholesterol, sphingomyelin, and vitamin E. 

Hauk PJ, Goleva E, Strickland I, et al. (2002) Increased glucocorticoid receptor Beta expression converts mouse hybridoma cells to a corticosteroid-insensitive phenotype. Am J Respir Cell Mol Biol. 27,361-367. 

Depending on the starting material, preantibody incubation steps may vary and are outlined in Chapters 9-12. The following assay begins with the removal of the slides from the overnight 10% normal serum incubation step. Since the ABC technique is universal in application, and a biotinylated secondary antibody exists for virtually any primary antibody, this protocol will assume a monoclonal assay and a primary antibody from a mouse hybridoma. A good secondary antibody to use in this situation is a biotinylated antimouse antibody made in horse. Therefore, the overnight serum incubation in this case would have been with 10% normal horse serum in PBS (see Note 4). 

Fusion with the cells compensates for this deficiency. When fused and unfused cells are incubated in the presence of the folic acid antagonist aminopterin, the de novo synthesis of purines and pyrimidines for DNA is blocked. Cells deficient in HGPRT die, whereas hybrid cells are able to bypass aminopterin blockage by metabolism of hypoxanthine and thymidine added to the medium. In the generation of mouse hybridomas, an number of myelomas deficient in HGPRT are available, all originating from MOPC 21, a spontaneous myeloma from the BALB/c mouse strain. 

Mouse hybridomas growing in the MiniPERM can be rotated at speeds of 5-20 rpm. Cells sensitive to shearing forces should be turned at lower rpm. 

The reduced glycolytic rate and formation of lactic acid may result in a slower growth rate (Griffiths, 2000). Duval et al. (1992) cultured a mouse hybridoma cell line (VO 208) in batch/fed-batch cultures in a medium supplemented with fructose instead of glucose and demonstrated an increase of the lifespan of the culture and an enhancement in the antibody secretion. However, it has been reported that complete glucose substitution by other hexoses can alter product glycosylation (Paredes et al., 1999). 

Duval D, Demangel C, Munier-Jolain K, Miossec S, Geahel I (1991), Factors controlling cell proliferation and antibody production in mouse hybridoma cells I. Influence of the amino acid supply, Biotechnol. Bioeng. 38 561-570. 

ELISA, you will determine the titer of this antibody, as well as that of a monoclonal antibody against /3-galactosidase, called J1, that is derived from a mouse hybridoma cell line. Based on the titer of the J1 antibody, you will perform a competitive ELISA to determine the concentration of /3-galactosidase in an unknown solution. 

Boraston R, Garland S Birch JR (1983) Growth and antibody production by mouse hybridoma cells growing in oxygen-limited chemostat culture. Journal of Chemical Technology and Biotechnology 33B 200. 

Boraston R, Thompson PW, Garland S Birch JR (1984) Growth and oxygen requirements of antibody producing mouse hybridoma cells in suspension culture. Developments in Biological Standardisation 55 103-111. 

The mouse-mouse hybridoma CRL-1606 cell line producing IgGl monoclonal antibody (MAb) against human fibronectin was obtained from ATCC. Batch cultures... 

Mechetner E. Development and Characterization of Mouse Hybridomas Monocolonal Antibodies. Ed. Maher Albitar. SpringerLink New York, 2007, pp. 1-13. 

Favre (1993) found that his data for constant volume perfusion of two different mouse-mouse hybridoma lines could not fit the classical constant volume filtration equation. He suggested that this could be due to a monolayer type of cell buildup instead of a cake in the conventional filtration. According to Favre, a model principally based on stochastic and steady plugging of the pores in the screen by the cells leading to an equilibrium state can possibly give a rational design procedure for spin filters. 

Oh SKW, Nienow AW, Al-Rubeai M, Emery AN. (1992) Eurther studies of the culture of mouse hybridomas in an agitated reactor with and without continuous sparging. 

Lymphocytes Human + Myeloma Hybridoma Mouse Hybridoma morphol. Berg 1982... 

The fusion cultures are examined for the growth of hybridoma colonies from seven days onwards. Mouse hybridomas tend to grow initially as discrete colonies (sphaeroids) whereas hybridomas produced with the Y3 myeloma are quite diffuse and not easy to recognize. With further growth the Y3 hybridomas form discrete colonies that can be quite firmly attached to the plastic. Screening is normally carried out initially at 10-14 days post-fusion by removing samples of about 200 pi of supernatant. 

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