Theproductionofmonoclonalantibodiesinvolvesthefusionofmyelomacellswithspleencellsandtheselectionofhybridomas(cellhybrids)incultureutilizinghypoxanthine-aminopterin-thymidine(HAT)supplementedmedium.AminopterinblocksthesynthesisofDNAbyinhibitingdihydrofolatereductaseCellsthatlacktheABIlitytoutilizethesalvagepathwayfornucleotidesynthesisareeliminated.Cellsthatpossesshypoxanthine-guaninephosphoribosyltransferase(HPRTase)andthymidinekinase(TK)enzymescanutilizethesalvagepathwayifsuppliedwithhypoxanthineandthymidine.||Thepurposeofthemediumisto:(1)selectivelykillunfusedmyelomacellsthatarewelladaptedtotissuecultureandwouldotherwiseoutgrowanyhybridomasproducedand(2)eliminateanymyeloma-myelomahybridomasthatlackHPRTase.HPRTasepositivespleen-spleenhybridomas,althoughnotsensitivetoaminopterin,arenormallyshort-livedinculture.||Afterselectioniscomplete(~10-14weeks),aminopterinisdilutedfromtheculturebyseveralpassagesofthecellsinhypoxanthine-thymidine(HT)supplementedmedium(~2-3weeks)beforetransferintonormalhybridomagrowthmedium.||Form:|Whitetooff-whitepowder||Solubility(in10mlwater):|Colorlesstofaintyellow,clearliquid||Sterility:|Sterile(gamma-irrADIated)||Endotoxin:|≤100EU/ml||Components:|Eachvialcontains:|Hypoxanthin:6.8mg|Aminopterin:0.088mg|Thymidine:1.94mg||PreparationInstructions:|Reconstitutevialto10mlwithsterilewatertoyielda50Xsolutioncontaining0.005Mhypoxanthine,0.00005Maminopterinand0.0008Mthymidine.When10mlof50Xconcentratearedilutedto500mlwithsteriletissueculturemedium,thefinalconcentrationsofhypoxanthine,aminopterinandthymidineare100uM,0.4uMand16uM,respectively.||StorageandStability:|Storelyophilizedproductat-20°Cpriortoreconstitution.Lyophilizedpowderisstablefor6monthsat20°Cafterreceipt.Uponreconstitutionto10ml,theconcentratedstockcanbestoredat-20°C.

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