2. Cell fusion
Thoroughly clean lymphocytes are blend with HGPRT deteriorative myeloma cells in this stage . Next the combination of the cells are disclosed to a strong concentration of PEG and unification is occur. The PGE Chuck out by washing and the cells are maintained In a well nurse medium. Three types of cells are found in the medium. They are –

? combination of Hybridomas
?individual myeloma cells
?individual lymphocytes

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3. Selection
HAT medium ( hypoxanthine aminopterin thymidine medium) is usually used for the selection of hybrid cells. In HAT medium the cellular synthesis of purines and pyrimidines from simple sugar is blocked by aminopterin. But some cells can flourish by taking advantage of hypoxanthine and thymidine existent in the medium by salvage pathway utilizing hypoxanthine guanine phosphoribosyl transferase (HGPRT). But HGPRT is deficient in myeloma cells. So when aminopterin block denovo pathway myeloma cells can not survive in HAY medium. Beta cell can survive in the HAT medium as they are HGPRT+ . Beta cell go through instinctive cell death following some division.