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The assay for PMA/ionomycin-stimulated PBMC proliferation was performed in 96-well flat-bottomed microtitre plates. Compounds were prepared as 10 mM stock solutions in dimethyl sulfoxide. A 50-fold dilution of this was prepared in RPMI and serial dilutions were prepared from this solution 10 μl of the 50-fold diluted stock, or dilutions of it, were added to the well to give concentrations in the assay starting at 9.5 μM and going down. Into each well was placed 1×105 PBMC, prepared from human peripheral blood from a single donor, in RPMI1640 medium supplemented with 10% human serum, 2 mM glutamine and penicillin/streptomycin. Phorbol myristate acetate (PMA) (0.5 ng/ml final concentration) and ionomycin (500 ng/ml final concentration) were added to these cells in supplemented RPMI1640 medium (as above) so that the final volume of the assay was 0.2 ml. The cells were incubated at 37° C. in a humidified atmosphere at 5% carbon dioxide for 72 hours. 3H-Thymidine (0.5 μCi) was added for the final 6 hours of the incubation. The level of radioactivity incorporated by the cells was then determined and this is a measure of proliferation.

1. customA 50-fold dilution of this was prepared in RPMI and serial dilutions
2. additionwere added to the well
3. customto give
4. concentrationconcentrations in the assay
5. customprepared from human peripheral blood from a single donor, in RPMI1640 medium
6. concentrationPhorbol myristate acetate (PMA) (0.5 ng/ml final concentration) and ionomycin (500 ng/ml final
7. concentrationconcentration)
8. additionwere added to these cells in supplemented RPMI1640 medium (as above) so that the final volume of the assay
9. addition3H-Thymidine (0.5 μCi) was added for the final 6 hours of the incubation