The optimization of the mouse lymphocyte Hprt mu-included Con A, phytohemagglutinin, and a caltation assay has been impeded by the relatively cium ionophore ionomycin combined with a tumor poor growth potential of mouse T-cells in vitro, promoter phorbol 12-myristate 13-acetate. In a piwhich leads to low cloning efficiencies (CEs) and lot study, stimulation with Con A proved to be the limited expansion of Hprt mutant clones for molecu-most effective means for propagating mouse T-cell lar analysis of mutations occurring in control and clones under the various conditions tested. In foltreated mice. In this study, the addition and manipu-low-up experiments, transfer of mutant clones to lation of concanavalin A (Con A), mouse interleukin-24-well plates and repeated stimulation with Con 2 (IL-2), and a commercially available culture sup-A in IL-2 and rat T-STIM TM supplemented medium plement, rat T-STIM TM with Con A, were used to was found to expand 76% of 536 mutant clones to identify growth conditions producing relatively high about 400,000 to several million cells per clone. CEs for mouse T-cells. Supplementation of medium These data indicate that rat T-STIM TM -supplewith 10% rat T-STIM TM , along with appropriate mented medium enhances the initial outgrowth of amounts of Con A for priming and exogenous IL-2 mouse T-cells, and that repeated mitogenic stimufor cloning, resulted in average CEs of 15 -16% in lation with Con A in the presence of IL-2 and rat lymphocytes isolated from spleens of control mice T-STIM TM provides a means for propagating mouse (n Γ
32) or mice exposed to 1,3-butadiene (n Γ
T-cell clones for mutation analyses by a variety of 27). In addition, several reagents were assessed methods.