Dexamethasone Effects on Hybridoma Formation

To assess the effects of sterioids on the formation of hybridomas, sheep red blood cell (SRBC) immunized spleen cells from Balb/c mice were fused with P3U1 myeloma cells. After fusion, cells were grown for 1 week in HAT medium containing 10-3, 10-5, 10-7, 10-9 mM dexamethasone (DM) or HAT alone. Subsequently spent media was removed and refeeding with HAT was commenced. Parameters measured were: clone number, size, antibody production and DNA content. At concentrations of DM given above, the number of clones produced was 16, 39, 24, 29 respectively as compared to 40 clones in control wells. At 10-3 and 10-5 mM DM parental cells were markedly suppressed. All clones were < 1 mm in size at 10-3 mM, whereas at 10-5 mM and in controls, 43% and 46% of the clones respectively were 1–4 mm. By micro hemagglutination it was found that only 5% of the clones at 10-3 mM and 10% at 10-5 mM produced anti-SRBC antibody. In contrast, at lower doses and in control plates 99% of clones were antibody producers. Computerized DNA-RNA flow cytometry using acridine orange indicated that DNA loss was more pronounced in clones exposed to DM than controls. This method also indicated a lower number of parental cells in cultures grown in the presence of DM.