The production of monoclonal antibodies involves the fusion of myeloma cells with spleen cells and the selection of hybridomas (cell hybrids) in culture utilizing hypoxanthine-aminopterin-thymidine (HAT) supplemented medium. Aminopterin blocks the synthesis of DNA by inhibiting dihydrofolate reductase Cells that lack the ability to utilize the salvage pathway for nucleotide synthesis are eliminated. Cells that possess hypoxanthine-guanine phosphoribosyl transferase (HPRTase) and thymidine kinase (TK) enzymes can utilize the salvage pathway if supplied with hypoxanthine and thymidine.
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. HPRTase positive spleen-spleen hybridomas, although not sensitive to aminopterin, are normally short-lived in culture.
After selection is complete (~10-14 weeks), aminopterin is diluted from the culture by several passages of the cells in hypoxanthine-thymidine (HT) supplemented medium (~2-3 weeks) before transfer into normal hybridoma growth medium.
Form:White to off-white powder
Solubility (in 10ml water):Colorless to faint yellow, clear liquid
Preparation Instructions:Reconstitute vial to 10ml with sterile water to yield a 50X solution containing 0.005M hypoxanthine, 0.00005M aminopterin and 0.0008M thymidine. When 10ml of 50X concentrate are diluted to 500ml with sterile tissue culture medium, the final concentrations of hypoxanthine, aminopterin and thymidine are 100uM, 0.4uM and 16uM, respectively.
Storage and Stability:Store lyophilized product at -20°C prior to reconstitution. Lyophilized powder is stable for 6 months at 20°C after receipt. Upon reconstitution to 10ml, the concentrated stock can be stored at -20°C.
