반응 #833838

반응 방정식

penicillin streptomycin

PMA ionomycin

glutamine

→

Ionomycin

용매

dimethyl sulfoxide

반응 조건

후처리

1. 1
   기타A 50-fold dilution of this was prepared in RPMI and serial dilutions
2. 2
   workup.ADDITIONwere added to the well
3. 3
   기타to give
4. 4
   농축concentrations in the assay
5. 5
   기타prepared from human peripheral blood from a single donor, in RPMI1640 medium
6. 6
   농축Phorbol myristate acetate (PMA) (0.5 ng/ml final concentration) and ionomycin (500 ng/ml final
7. 7
   농축concentration)
8. 8
   workup.ADDITIONwere added to these cells in supplemented RPMI1640 medium (as above) so that the final volume of the assay
9. 9
   workup.ADDITION3H-Thymidine (0.5 μCi) was added for the final 6 hours of the incubation

실험 절차

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.

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