What concentration of brefeldin A to use?

Reagent Stock Concentration Final Concentration
Brefeldin A 5 mg/mL in DMSO (store in aliquots at -20C) 10 μg/mL (1:50) or 5μg/mL (1:100) with monensin
Monensin 5 mg/mL in ethanol (store at -20C) 10 μg/mL (1:50) or 5 μg/mL (1:100) with brefeldinA
Anti-CD3/ CD28 Follow manufacturer instruc

Can you fix intracellular staining?

Fixation. If staining intracellular antigens (e.g. IFN-γ or IL-4), first perform cell surface antigen staining as described in BioLegend’s Cell Surface Immunofluorescence Staining Protocol, then fix cells in 0.5 ml/tube Fixation Buffer in the dark for 20 minutes at room temperature.

What is intracellular staining?

Intracellular cytokine staining (ICS) is a method of cytokine analysis that provides information on the type of cytokines that are produced upon (antigen-specific) stimulation of T cells and B cells.

What is intracellular cytokine staining used for?

Intracellular cytokine staining (ICS) is a very useful and widely used flow cytometry based assay which detects the production and accumulation of cytokines within the endoplasmic reticulum after cell stimulation.

How do you dilute brefeldin A?

Formulation: Brefeldin A Solution is supplied as a 1000X in DMSO, which should be diluted to 1X in cell culture medium. 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.

How does PMA ionomycin work?

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.

Can you permeabilize cells without fixing?

Hi Husne, just to add to the great answers above, when you permeabilize cells without fixation all small soluble cytoplasmic proteins will eventually diffuse out of the cell. The smaller the cytoplasmic protein is, the faster it will be lost without fixation.

Do you need to fix cells for intracellular staining?

To stain intracellular molecules, the cells need to be fixed in suspension and then permeabilized before the detection antibody is added.

How do you dissolve ionomycin?

Add 100 μl of DMSO (not provided) to 1 mg of ionomycin. Mix by vortexing until completely dissolved.

What is PMA stimulation?

Mechanistically, PMA/ionomycin provides a potent stimulation allowing us to bypass the T cell receptor activation essential for Treg cell development and it prevents the emergence of CD4-CD8+ cells in the culture [17]. PMA activates protein kinase C [38] while ionomycin is a Ca2+ mobilizing agent [39].