1. Four female Balb-c mice (see Note 5).
3. Media: basal RPMI 1640 medium requires supplementation with sodium pyruvate, L-glutamine, and penicillin/streptomycin before use. The supplements are supplied as concentrates of 50X or 100X, and the appropriate amounts should be added. Standard tissue culture media for hybridoma production contain 5%, 10%, or 15% fetal bovine serum (FBS; see Note 6). Sufficient quantities should be prepared in advance and a sterility check should be performed on them prior to use.
All prepared media should be stored at -20°C until required and used within 28 d of thawing. L-Glutamine has an effective lifespan of about 28 d in prepared medium and will degrade giving rise to ammonia after this time.
Allogeneic mixed thymocyte medium (4) is a conditioned medium used to aid cells recover from the fusion process. It also encourages division of hybridoma cells after dilution cloning when colonies derived from single cells are required. Rat thymi are used for this purpose to minimise the number of animals required to produce adequate numbers of cells. The medium contains a number of "helper" cytokines produced by the thymocytes. It is important that the thymocytes used are derived from two different rat strains as this causes co-stimulation and enhanced cytokine secretion by the cells. Mixed thymocyte medium must always be diluted with RPMI 1640 medium containing 15% FBS and is usually used at a dilution of 10-15%.
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