Phorbol 12-myristate 13-acetate Molecular Biology Reagent Protocol

Product No. P1585

CAS RN 16561-29-8
Synonyms: PMA, TPA

Product Description

T-cell activation is normally triggered by the interaction of a cell surface receptor to its specific ligand molecule. This binding event triggers the rapid hydrolysis of inositol phospholipids to diacylglycerol and inositol phosphates by phospholipase C (PLC). Diacylglycerol is an allosteric activator of protein kinase C (PKC) activation and inositol phosphates, which trigger Ca++ release and mobilization, resulting in a cascade of additional cellular responses mediating T-cell activation. One of these cellular responses is the production and secretion of interleukin-2 (IL-2). Phorbol 12-myristate 13-acetate, which has a structure analogous to diacylglycerol, can also activate PKC.

Jurkat cells are a leukemic T-cell line known to produce IL-2. Under normal growth conditions, little to no IL-2 is produced in Jurkat cells. PMA, through its activation of PKC, can activate T-cells and stimulate low-level production of IL-2. When Jurkat cells are stimulated by PMA and a co-stimulator, such as PHA, IL-2 production is strongly enhanced2. Phytohemagglutinin can trigger a low level of T-cell activation and IL-2 production by binding non-specifically to the cell surface receptor complex. The combination of PMA and PHA results in greatly increased IL-2 production and secretion.

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Storage/Stability

Store product below 0 °C. All stock solutions should be stored at –20 °C. Protect from light.

Product Profile

Soluble in ethanol and DMSO.

Suitability Assay: Tested using Jurkat cells grown in the presence of 1 µg/mL phytohemagglutinin (PHA) and 50 ng/mL PMA. IL-2 production was >= 15,000 pg/106 Jurkat cells.

Procedure

2.5 mL of Jurkat cells (1 x 106 cells/ mL) and 2.5 mL of fresh media (RPMI 1640 + 10% fetal calf serum containing 10 mL/L penicillin-streptomycin) were added to 25 cm2 culture bottles. The following additions were made in duplicate.
a. Control - no additions
b. 1 µg/mL PHA Control - add 10 µL PHA stock solution (0.5 mg/mL PHA in filter-sterilized PBS)
c. 1 µg/mL PHA + 50 ng/mL PMA – 10 µL PHA stock solution + 2.5 ìl PMA stock solution (100 µg/mL PMA in DMSO)

The bottles were incubated at 37 °C for 24 hours. After centrifugation, the clarified broth was tested for IL-2 production using a Human Interleukin-2 ELISA test kit. The PMA + PHA test cultures yielded a level of production of IL-2>= 15,000 pg IL-2/106 Jurkat cells. The PHA control culture yielded <3,000 pg IL-2/106 Jurkat cells. The control with no addition was <50 pg IL-2/106 Jurkat cells.

Materials
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References

1.
Weiss Aea. 1984. The role of T3 surface molecules in the activation of human T cells: a two-stimulus requirement for IL 2 production reflects events occurring at a pre-translational level.. J. Immunol., . 133(1):123-128.
2.
Manger B, Hardy KJ, Weiss A, Stobo JD. 1986. Differential effect of cyclosporin A on activation signaling in human T cell lines.. J. Clin. Invest.. 77(5):1501-1506. http://dx.doi.org/10.1172/jci112464