Clonal stability and heterogeneity of hybridomas: analysis by multiparameter flow cytometry

Flow cytometry of cellular DNA and RNA content was employed to determine DNA ploidy, proliferation, and RNA content of hybridoma cultures shortly after cell fusion and sequentially thereafter. The parental mouse myeloma cell line P3UI was characterized by tetraploid DNA content, S-phase 50%, and high RNA, the parental mouse spleen cells by diploid DNA content, low proliferation, and low RNA content. Hybridoma cultures studied as early as 21 days after fusion were found to contain the sum of the parental cells' DNA content (hexaploid), or if less, more than that of the myeloma parental cells. Only one clone of 35 tested was shown to be hexaploid and the rest hypertetraploid or hyperpentaploid. Hybrid cell cultures were frequently found to contain a variable mixture of unfused parental cells. The high proliferation of hybridoma cells determined by flow cytometry indicates that these cells would eventually overgrow the parental cells. Flow cytometry also enabled an accurate estimation of the effect of various doses of dexamethasone added to HAT medium immediately after cell fusion on hybridoma formation. Cultures treated with 10(-5) mM of the hormone had a higher DNA ploidy than cultures grown in the presence of 10(-3) mM dexamethasone. No parental cells were observed in the hybridoma cultures studied with this hormone. Sequential DNA/RNA measurements of hybridoma clones showed a decrease in DNA ploidy over time with high dexamethasone doses and a minimal increase or no change with low hormone dose. Flow cytometry is suggested to be a useful technique for evaluating the effects of various agents on DNA ploidy and proliferation and on stability of fused cells.