HAT medium contains:
HAT medium intelligent metabolic selection exploiting two distinct pathways nucleotide biosynthesis de novo synthesis requiring tetrahydrofolate one-carbon donor and salvage reusing preformed purines pyrimidines. Aminopterin potent folic acid analog competitively inhibits dihydrofolate reductase converting dihydrofolate tetrahydrofolate Ki 0.03 nM blocking regeneration tetrahydrofolate required thymidylate synthase converting dUMP dTMP purine enzymes GAR transformylase AICAR transformylase shutting de novo purine thymidine synthesis hours. Cell survival then depends entirely salvage enzymes HPRT converting hypoxanthine supplied IMP thymidine kinase phosphorylating thymidine TMP. Myeloma partners deliberately selected HPRT-negative mutants via resistance 8-azaguanine analog kills HPRT-proficient cells incorporating toxic nucleotide cannot utilize hypoxanthine therefore die aminopterin presence 48h. Primary B cells naturally expire short lifespan 3 days. Only heterokaryons inheriting functional HPRT gene B cell immortal proliferation program myeloma proliferate forming visible colonies 10-14 days post fusion counted inverted microscope. Optimized formulation hypoxanthine 100 uM aminopterin 0.4 uM thymidine 16 uM RPMI 1640 20 percent FBS supporting selective growth hybridomas.
Ref: Littlefield Science 1964 HAT Selection Classic; Janeway Immunobiology HAT Composition Components Chapter 2; Lodish Cell Culture HAT Selection Principle Media.