INTRACELLULAR FLOW

Various activated cell types can secrete cytokines, chemokines, and other inflammatory mediators such as perforin and granzymes as part of an immune or inflammatory response.

Methods such as ELISA and BD™ Cytometric Bead Array (CBA) measure secreted proteins produced by entire cell populations. In contrast, intracellular flow cytometry allows the analysis of cytokines and other inflammatory mediators produced by individual, phenotypically identified cell types within cell populations of interest.


Intracellular flow cytometry makes it possible to easily determine if the cytokine production by an activated cell population is the result of a few cells producing large amounts of cytokine or a large population of cells producing small quantities of cytokine per cell. Moreover, intracellular flow cytometry makes it possible to easily measure multiple cytokines simultaneously, to identify polyfunctional cells.1 Intracellular cytokine staining is also useful for a variety of studies including B- and T-cell differentiation and plasticity.

Since cytokines typically are secreted proteins, they must first be trapped inside the cell by using a protein transport inhibitor. The two most commonly used protein transport inhibitors are monensin (BD GolgiStop) and brefeldin A (BD GolgiPlug). Monensin prevents protein secretion by interacting with the Golgi transmembrane Na++/H+ transport, while brefeldin A redistributes intracellularly produced proteins from the cis/medial Golgi complex to the endoplasmic reticulum.2 As a result, the best choice of protein transport inhibitor varies by cytokine and by species. See Table 1.


Species Cytokines Transport Inhibitor
Human IL-1α, IL-6, IL-8, TNF-α Monensin
Human IFN-γ, IL-2, IL-10, IL-12, MCP-1,
MCP-3, MIG, MIP-1α, RANTES
Either Monensin or Brefeldin A
Mouse IL-6, IL-12, TNF-α Brefeldin A
Mouse GM-CSF, IL-3, IL-4, IL-5, IL-10 Monensin
Mouse IFN-γ, IL-2 Either Monensin or Brefeldin A

IFN-γ and IL-2 production in CD8+ cells

IFN-γ and IL-2 production in CD8+ cells

PBMCs were stimulated with staphylococcal enterotoxin B for 6 hours in the presence of brefeldin A. After stimulation, cells were fixed and permeabilized using the BD Cytofix/Cytoperm buffer system. Cells were stained with the following fluorescent antibodies: CD3 FITC, CD4 PerCP-Cy™5.5, CD8 Brilliant Violet 421, IFN-γ PE, and IL-2 APC. Cells were then washed. Finally, cells were analyzed using a BD FACSVerse™ flow cytometer.


IFN-γ and IL-2 production in CD8+ cells.

PBMCs were stimulated with staphylococcal enterotoxin B for 6 hours in the presence of brefeldin A. After stimulation, cells were fixed and permeabilized using the BD Cytofix/Cytoperm buffer system. Cells were stained with the following fluorescent antibodies: CD3 FITC, CD4 PerCP-Cy™5.5, CD8 Brilliant Violet 421, IFN-γ PE, and IL-2 APC. Cells were then washed. Finally, cells were analyzed using a BD FACSVerse™ flow cytometer.

BD has simplified the detection of cytokines and cell surface markers with well established kits, buffer systems, and rich panels of fluorescent antibodies. Antibodies to surface markers and cytokines conjugated to a variety of fluorochromes are available. This allows for flexibility in staining panel design, supporting high content multicolor flow cytometric analyses to gain the most data from precious cell samples.

BD FastImmune™ kits contain tested cocktails of fluorescent antibodies, an appropriate protein transport inhibitor, compatible buffers, and a detailed protocol for the optimal preparation, staining, and detection of cytokine-producing cells from whole blood. Onboard assays on the BD FACSVerse flow cytometer system further simplify the process.

Cytokine - BD FastImmune CD4 intracellular cytokine detection Kit

BD Cytofix/Cytoperm buffer has been cited in thousands of publications for the analysis of cytokine-producing cells by flow cytometry. Researchers select the protein transport inhibitor best suited to their cytokine of interest and use the BD Cytofix/Cytoperm buffer system to fix, permeabilize, and stain their cells for flow cytometric analysis.



Antigen-specific IFN-γ production

Antigen-specific IFN-γ production by cytomegalovirus (CMV) pp65- stimulated CD4+CD69+ T lymphocytes

Two-color flow cytometric dot plots show IFN-γ vs CD69 expression by CD4 T cells that were either unstimulated (left panels), SEB-stimulated (as positive controls, center panels), and CMV pp65-stimulated (right panels) samples from two donors. Human whole blood was stimulated in the presence of brefeldin A before fixing, permeabilizing, and staining using the BD FastImmune™ 3-color CD4 intracellular cytokine detection kit. Data was acquired using a BD FACSVerse flow cytometer and a BD FACSuite™ software research assay.

 

Antigen-specific IFN-γ production by cytomegalovirus (CMV) pp65- stimulated CD4+CD69+ T lymphocytes

Two-color flow cytometric dot plots show IFN-γ vs CD69 expression by CD4 T cells that were either unstimulated (left panels), SEB-stimulated (as positive controls, center panels), and CMV pp65-stimulated (right panels) samples from two donors. Human whole blood was stimulated in the presence of brefeldin A before fixing, permeabilizing, and staining using the BD FastImmune™ 3-color CD4 intracellular cytokine detection kit. Data was acquired using a BD FACSVerse flow cytometer and a BD FACSuite™ software research assay.


1 Seder RA, Darrah PA, Roederer M. T-cell quality in memory and protection: implications for vaccine design. Nat Rev Immunol. 2008;8:247-258.

2 Schuerwegh AJ, Stevens WJ, Bridts CH, De Clerck LS. Evaluation of monensin and brefeldin A for flow cytometric determination of interleukin-1 beta, interleukin-6, and tumor necrosis factoralpha in monocytes. Cytometry. 2001;46:172-176.