Cell Stimulation Reagent (without Brefeldin A) 500x is a mixture of Phorbol-12-myristate-13-acetate (PMA) and ionomycin. PMA activates protein kinase C and ionomycin acts as a Ca2+ ionophore allowing movement of Ca2+ across membranes. Activation of cells with PMA and ionomycin leads to an increase in surface expression of certain markers such as CD69 and CD154 and the production of cytokines such as IL-2, IFN-gamma and TNF-alpha.
- Product Form
- 500x concentrate - liquid
- 500x solution contains: Phorbol-12-myristate-13-acetate (PMA) 40.5 μM and Ionomycin 669.3 μM in DMSO
- Store at -70oC.
Storage in frost-free freezers is not recommended.
This product should be stored undiluted. This product is photosensitive and should be protected from light.
- Guaranteed until date of expiry. Please see product label.
- For research purposes only
This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visit the antibody protocols page.
Applications of Cell Stimulation Reagent (Without Brefeldin A)
Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own system using appropriate negative/positive controls.
- Instructions For Use
- 1. Resuspend 1 x106 - 2 x106 cells per ml in cell culture media
2. Thaw the Cell Stimulation Reagent (without Brefeldin A) 500x (BUF076A) in a 37oC water bath
3. To each ml of cell suspension add 2 μl of BUF076A and incubate cells in a CO2, 37oC incubator for 6 hours
4. Harvest cells and centrifuge at 300-400 g for 5 minutes. Discard supernatant
5. To wash the pellet, add 10-20 ml of Cell Staining Buffer (BUF073), vortex to resuspend the pellet then centrifuge at 300-400 g for 5 minutes. Discard supernatant and repeat step 5 for a second time
6. Stain for surface markers or proteins of interest and analyze cells using flow cytometry
Note: Aliquot product to avoid repeat freeze-thawing. Time and culture conditions may need to be optimized. To detect intracellular staining following activation, the Cell Activation Reagent (without Brefeldin A) 500x (BUF076A) can be used with Monensin Solution (BUF074) or Brefeldin A Solution 1000x (BUF075).
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