Cell Stimulation Reagent (with Brefeldin A)
Cell Stimulation Reagent (with Brefeldin A) 500x is a mixture of PMA (phorbol-12-myristate-13-acetate), ionomycin and the protein transport inhibitor Brefeldin A. PMA and ionomycin activate cells and increase cytokine production. Brefeldin A allows for these cytokines to accumulate in the rough endoplasmic reticulum and golgi apparatus allowing for their detection by intracellular fluorescent staining.
- Product Form
- 500x concentrate - liquid
- 500x solution contains: PMA (phorbol-12-myristate 13-acetate) 40.5 μM, Ionomycin 669.3 μM and Brefeldin A 2.5 mg/ml 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.
- Shelf Life
- Please see label for expiry date.
- For research purposes only
Applications of Cell Stimulation Reagent (with Brefeldin A)
|Application Name||Verified||Min Dilution||Max Dilution|
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 (with Brefeldin A) 500x (BUF077A) in a 37oC water bath
3. To each ml of cell suspension add 2 μl of BUF077A 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 by flow cytometry
Note: Aliquot product to avoid repeat freeze-thawing. Time and culture conditions may need to be optimized.
Activation of cells for >12 hours with Brefeldin A may affect cell viability. In these circumstances it is recommended to use Cell Stimulation Reagent (without Brefeldin A) 500x (BUF076A) and then add Brefeldin A Solution 1000x (BUF075) or Monensin Solution (BUF074).
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Useful Reagents Available
|Description||Product Code||Pack Size||Applications||List Price||Quantity|
|Monensin Solution (1000X)||BUF074||1 ml||F|
|Brefeldin A Solution (1000X)||BUF075||1 ml||F|
|Cell Stimulation Reagent (Without Brefeldin A) (500X)||BUF076A||0.1 ml||F|