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 HP
Ref: Littlefield Science 1964 HAT Selection Classic; Janeway Immunobiology HAT Composition Components Chapter 2; Lodish Cell Culture HAT Selection Principle Media.