The BD FastImmune Cytokine Dectection System is a method for detecting intracellular cytokine production using flow cytometry. The protocol involves fixing and permeabilizing the cells of interest and then staining for surface markers and cytokine(s) using fluorescently labeled antibodies. The samples are then analyzed by flow cytometry. This method has many potential uses and can produce useful cytokine profiles from a mixture of cell types including whole blood.
I use the FastImmune products on a regular basis. Primarily I use it to analyze cytokine production in human T cells as well as other human blood cells. In a typical experiment, I stain stimulated T cells with fluorescently conjugated anti-IFNg, to determine antigen specificity for a given human donor, and frequently look at TNFa as well. In conjunction with the cytokine antibodies, I also use an antibody cocktail for T cell surface markers including CD69 (a T cell activation marker), CD4 and CD8. Using four-color flow cytometry, I have been able to identify specific CD4 and CD8 populations of cytokine producing cells from stimulated T cells. I typically detect a range of responses from 0.1% to 1-2% of CD4 or CD8 cells as cytokine positive. I have also used this system to determine the specificity and function of T cell clones. I have not done whole blood intracellular staining or used the system with mouse cells, though both are possible.
The biggest advantage to this protocol is that it allows the detection of cytokine production from a specific cell type since you can detect surface markers of the producing cells. Using kits that detect a cytokine that is secreted by cells does not identify the phenotype of the active cell. Another advantage is the cytokine positive cells can be sorted using flow cytometry.
One of the disadvantages of this assay is that it can be a technically challenging assay to perform. Being competent with flow cytometry and experienced with analyzing intracellular staining is extremely helpful. Further, while the cyokine positive cells can be sorted, they are fixed and permeabilized so they cannot be grown in culture. Since the cytokine producing populations are typically small, it is very important to have low background in the negative control so your positive populations are clearly positive.
In summary, the FastImmune cytokine detection system has the potential to provide valuable information on cytokine production from a mixture of cells such as peripheral blood mononuclear cells or whole blood. If done on a routine basis it is worthwhile to tackle the technical aspects of this assay, but if you are planning on using this product for a one-time experiment, you would probably be better off collaborating with someone who has experience with it already.
Amy Hobeika, PhD
Postdoctoral Associate
Duke University Medical Center