What is PMA ionomycin?

Published by Charlie Davidson on

What is PMA ionomycin?

PMA activates protein kinase C, while ionomycin is a calcium ionophore, and stimulation with these compounds bypasses the T cell membrane receptor complex and will lead to activation of several intracellular signaling pathways, resulting in T cell activation and production of a variety of cytokines.

What is PMA in flow cytometry?

While PMA (phorbol 12-myristate 13-acetate) and ionomycin (calcium ionophore) are often used in combination to induce cytokine production, more specific stimulation or cell-type activation of agonistic antibodies against cell receptors, such as CD3 and CD28 for T lymphocytes, are a great option.

How much brefeldin to add?

For most cytokines: use brefeldin A at 10 μg/mL final concentration (see stock preparation table). For CD107 and CD154: use monensin at 10 μg/mL final concentration (see stock preparation table).

How much does brefeldin cost?

Brefeldin A Solution (1,000X)

Cat # Size Price
420601 1 mL $55

What activates PMA?

PMA, through its activation of PKC, can activate T-cells and stimulate low-level production of IL-2. Phytohemagglutinin can trigger a low level of T-cell activation and IL-2 production by binding non-specifically to the cell surface receptor complex.

What is GolgiStop?

Description. The ex vivo addition of BD GolgiStop™, a protein transport inhibitor containing monensin, to in vitro- or in vivo-stimulated lymphoid cells blocks their intracellular protein transport processes. This results in the accumulation of cytokines and/or proteins in the Golgi complex.

How do you store brefeldin A?

Storage: Store the Brefeldin A Solution (1,000X) between 2°C and 8°C. Note: DMSO freezes at this temperature. Recommended Usage: Dilute the 1000X solution to 1X in the tissue culture medium. It is recommended that cells are cultured with brefeldin A for ≤ 24 hours, as this can become toxic for cell viability.

What does PMA do to 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.

How does PMA activate PKC?

PMA stimulates conventional (α, βI, βII, γ) and novel (δ, ε, η, θ) PKC by mimicking the activating ligand DAG[8]. PKC isoforms of all classifications have been reported in neutrophils from healthy donors[10].

How does Brefeldin A increase the production of cytokines?

Incubation of cells in culture with brefeldin A leads to blockade of protein transport to the Golgi complex (GC) and accumulation of proteins in the endoplasmic reticulum (ER). Addition of brefeldin A during the last hours of in vitro activation of cells results in enhanced detection of intracellular cytokines.

Which is a better protein transport inhibitor monensin or Brefeldin?

Brefeldin A was found to be a more suitable protein transport inhibitor than monensin. This method will be useful for analysing the nature of individual rat cytokine-producing cells in a variety of experimental model systems. Publication types

How to test for intracellular cytokine in PBMCs?

Add 1 μg/ml final concentration of CD28 and/or CD49d (labeled antibody can be used if analysis of the marker is desired). Incubate the cells for 4 h (PMA + ionomycin stimulation, PHA + ionomycin stimulation) or 6-8 h (SEB, anti-CD3/CD28 stimulation, peptide stimulation) at 37 °C, in a CO 2 incubator.

What is the dilution factor for vicell PBMC?

Load onto Vicell as PBMC with a 1:25 dilution factor. Adjust the cell concentration to 5-10 x 10 6 cells/ml with warm media (no more benzonase at this point). Using a multichannel pipette, add 200 µl cells (for at least 1 x 10 6 cells) into each well of a 96-well deep well plate.

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