Cell Stimulation Reagent (Without Brefeldin A)

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.

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.

Application Name	Verified	Min Dilution	Max Dilution
Flow Cytometry

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 37^oC water bath
3. To each ml of cell suspension add 2 μl of BUF076A and incubate cells in a CO₂, 37^oC 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).