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Cell Activation Cocktail (with Brefeldin A)

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Regulatory Status
RUO
Other Names
Cell Stimulation Cocktail, PMA/Ionomycin
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Product Citations
publications
Cell_Activation_Cocktail_with_Brefeldin_1_022814
Human peripheral blood lymphocytes were stimulated with Cell Activation Cocktail (with Brefeldin A) for 6 hours, fixed, permeabilized, and then stained with CD3 APC and TNF-α PE (top) or IFN-γ Brilliant Violet 421™ (bottom).
  • Cell_Activation_Cocktail_with_Brefeldin_1_022814
    Human peripheral blood lymphocytes were stimulated with Cell Activation Cocktail (with Brefeldin A) for 6 hours, fixed, permeabilized, and then stained with CD3 APC and TNF-α PE (top) or IFN-γ Brilliant Violet 421™ (bottom).
  • Cell_Activation_Cocktail_with_Brefeldin_2_022814
Cat # Size Price Quantity Check Availability Save
423303 100 µL 112 CHF
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423304 400 µL 335 CHF
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Description

Cell Activation Cocktail (with Brefeldin A) is a pre-mixed cocktail with optimized concentration of PMA (phorbol 12-myristate-13-acetate), ionomycin, and protein transport inhibitor (Brefeldin A). Activation of cells with PMA and ionomycin leads to cytokine production that is captured in the rough endoplasmic reticulum and Golgi apparatus by the protein transport inhibitor Brefeldin A. Brefeldin A functions to inhibit intracellular protein transport, thus allowing intracellular detection of the secreted cytokine and proteins by immunofluorescent staining.

Product Details
Technical Data Sheet (pdf)

Product Details

Formulation
Cell Activation Cocktail (with Brefeldin A) is composed of PMA, ionomycin, and Brefeldin A in DMSO (500X).
Concentration
Each vial of this cocktail contains phorbol-12-myristate 13-acetate (40.5 µM), ionomycin (669.3 µM), and Brefeldin A (2.5 mg/ml) in DMSO (500X).
Storage & Handling
Store cocktail at -70°C upon receipt. Protect from light. Avoid repeat freeze/thaw cycles.
Application

Activ, FC, ICFC - Quality tested
Recommended Usage

Cat. No. 423303 contains 100 µL of cocktail, formulated at 500X, that can be used for activating 50 mL of cells of various types.

Cat. No. 423304 contains 400 µL (4 vials x 100 µL per vial) of cocktail, formulated at 500X, that can be used for activating 200 mL of cells of various types.

Application Notes

Cell Activation Protocol:
1. Resuspend cells of interest in desired cell culture medium. Recommended cell suspension is between 1-2 x 106 cells/mL, although effective activation can be obtained with higher or lower concentration.
2. Completely thaw vials in 37°C water bath.
3. Add 2 µL of the cocktail to each mL of cell suspension.
4. Incubate cells at 37°C in a CO2 incubator for 6 hours or the time period of interest.
5. Harvest activated cells and centrifuge at 350g for 5 minutes. Discard supernatant.
6. Add 2.5 mL of Cell Staining Buffer (Cat. No. 420201) to the cell pellet, mix by vortex or pipetting, then centrifuge at 350g for 5 minutes. Discard supernatant.
7. Repeat step 6.
8. Cells are now ready to be tested for surface markers or proteins of interest.

Note: Activation of cells overnight (>12 hours) with Brefeldin A is generally not recommended due to some level of toxicity. Under these conditions, it is recommended to use Cell Activation Cocktail without Brefeldin A (Cat. No. 423301), and then add monensin (Cat. No. 420701) at appropriate concentrations.

This product is temperature sensitive; repeated freeze/thaw is not recommended.

Intracellular and surface flow staining, as well as ELISA, can be performed following activation of cells with this cocktail.

Product Citations
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Antigen Details

Gene ID
NA

Related FAQs

The Brefeldin A solution freezes at 4°C . Is the solution still OK?

We recommend to thaw in warm water after removing from the fridge.  The Brefeldin A is dissolved in DMSO, which freezes at 18°C, so this is normal.  No one has reported any problems with the freeze/thaw cycles and this is how we recommend it is stored.   If customers are concerned, they can aliquot the Brefeldin A to avoid the freeze thaw.

How do I choose monensin or Brefeldin A solution?

Generally, Brefeldin A is more toxic for longer term incubations, so for shorter stimulations (6 hrs or less) use Brefeldin A.  For longer stimulations use monensin.  We recommend optimization for each cell type and protocol.

Go To Top Version: 3    Revision Date: 07.03.2019

For Research Use Only. Not for diagnostic or therapeutic use.

 

This product is supplied subject to the terms and conditions, including the limited license, located at www.biolegend.com/terms) ("Terms") and may be used only as provided in the Terms. Without limiting the foregoing, BioLegend products may not be used for any Commercial Purpose as defined in the Terms, resold in any form, used in manufacturing, or reverse engineered, sequenced, or otherwise studied or used to learn its design or composition without express written approval of BioLegend. Regardless of the information given in this document, user is solely responsible for determining any license requirements necessary for user’s intended use and assumes all risk and liability arising from use of the product. BioLegend is not responsible for patent infringement or any other risks or liabilities whatsoever resulting from the use of its products.

 

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