Reaktion #218731

Reaktionsgleichung

PMA ionomycin

glutamine

penicillin streptomycin

→

ionomycin

Lösungsmittel

dimethyl sulfoxide

Reaktionsbedingungen

Aufarbeitung

1. 1
   SonstigeA 50-fold dilution of this was prepared in RPMI and serial dilutions
2. 2
   Sonstigewere prepared from this solution
3. 3
   workup.ADDITION10 μl of the 50-fold diluted stock, or dilutions of it, were added to the well
4. 4
   Sonstigeto give
5. 5
   Einengenconcentrations in the assay
6. 6
   Sonstigeprepared from human peripheral blood from a single donor, in RPMI1640 medium
7. 7
   EinengenPhorbol myristate acetate (PMA) (0.5 ng/ml final concentration) and ionomycin (500 ng/ml final
8. 8
   Einengenconcentration)
9. 9
   workup.ADDITIONwere added to these cells in supplemented RPMI1640 medium (as above) so that the final volume of the assay
10. 10
    workup.ADDITION3H-Thymidine (0.5 μCi) was added for the final 6 hours of the incubation

Vorschrift

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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