PHORBOL 12-MYRISTATE 13-ACETATE(4β9α12β13α20-pentahydroxytiglia-16-dien-3-one12β-myristate 13-acetate 12-O-tetradecanoylphorbol 13-acetate)Molecular Biology Reagent

Product No P 1585

Product DescriptionT-cell activation is normally triggered by the interactionof a cell surface receptor to its specific ligand molecule This binding event triggers the rapid hydrolysis ofinositol phospholipids to diacylglycerol and inositolphosphates by phospholipase C (PLC) Diacylglycerolis an allosteric activator of protein kinase C (PKC)activation and inositol phosphates which trigger Ca++

release and mobilization resulting in a cascade ofadditional cellular responses mediating T-cell activation One of these cellular responses is the production andsecretion of interleukin-2 (IL-2) Phorbol 12-myristate13-acetate which has a structure analogous todiacylglycerol can also activate PKC

Jurkat cells are a leukemic T-cell line known to produceIL-2 Under normal growth conditions little to no IL-2 isproduced in Jurkat cells PMA through its activation ofPKC can activate T-cells and stimulate low-levelproduction of IL-2 When Jurkat cells are stimulated byPMA and a co-stimulator such as PHA IL-2 productionis strongly enhanced2 Phytohemagglutinin can triggera low level of T-cell activation and IL-2 production bybinding non-specifically to the cell surface receptorcomplex The combination of PMA and PHA results ingreatly increased IL-2 production and secretion

StorageStabilityStore below 0 degCAll stock solutions should be stored at ndash20 degC

Product ProfileTested using Jurkat cells grown in the presence of1 microgml phytohemagglutinin (PHA) and 50 ngml phorbol12-myristate 13-acetate IL-2 production was pg106 Jurkat cellsSoluble in ethanol and DMSO

Suitability Assay25 ml of Jurkat cells (1 x 106 cells ml) and 25 ml offresh media (RPMI 1640 + 10 fetal calf serumcontaining 10 mll penicillin-streptomycin) were added to25 cm2 culture bottles The following additions weremade in duplicate

a Control - no additionsb 1 microgml PHA Control - add 10 microl PHA stock solution

(05 mgml PHA in filter-sterilized PBS)c 1 microgml PHA + 50 ngml PMA ndash 10 microl PHA stock

solution + 25 microl PMA stock solution (100 microgmlPMA in DMSO)

The bottles were incubated at 37 degC for 24 hours Aftercentrifugation the clarified broth was tested for IL-2production using a Human Interleukin-2 ELISA test kit(Sigma Stock No CKH-102) The PMA + PHA testcultures yielded a level of production of IL-2 pgIL-2106 Jurkat cells The PHA control culture yieldedlt3000 pg IL-2106 Jurkat cells The control with noaddition was lt50 pg IL-2106 Jurkat cells

References1 Weiss A et al J Immunol 133 123 (1984)2 Manger B et al J Clin Invest 77 1501 (1986)

JWM2002

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