Immunology for Non-Immunologists: Cytokine Measurement

What Are Cytokine Measurement Assays?

Cytokine measurement assays are sensitive measures for both primary cell assays and purified antigen-specific cultures. Detecting the presence of cytokines — small proteins that are secreted by immune cells to signal an immune or inflammatory response — can help researchers better understand how our bodies respond to invading pathogens.

Common cytokines you may want to measure for immunology research include:

• IFNγ
• TNFα
• Interleukin 1β (IL-1β)
• Interleukin 2 (IL-2)
• Interleukin 4 (IL-4)
• Interleukin 5 (IL-5)
• Interleukin 6 (IL-6)
• Interleukin 8 (IL-8)
• Interleukin 10 (IL-10)
• Interleukin 12p70 (IL-12p70)
• Interleukin 13 (IL-13)

There are two categories of measuring cytokine production: collection from culture medium and intracellular staining.

Learn more about each method below.

Collection from Culture Medium

It is possible to collect bulk medium from culture after allowing time for stimulation to take place. From there you can measure a single cytokine using the traditional ELISA method or with the help of advanced equipment such as Meso Scale or Luminex instruments, measure multiple cytokines using a relatively small sample volume.

Research has shown that multiplex assays are just as accurate as singular cytokine measurement assays with the added benefit of being more time- and cost-effective.

Whether you are measuring one or multiple cytokines, pay close attention to the timing of cytokine release to get the best data. When running a traditional ELISA, it is possible to completely miss a cytokine if you sample too early or too late. Start by understanding the optimal measurement time point for your cytokine of interest and capture data accordingly to get the best readouts.

Figure 1 shows sample data from antigen-specific T cells that respond to tetanus toxoid. We stimulated the tetanus toxoid-specific T cells with peptide-pulsed dendritic cells and measured for the presence of ten common cytokines. The colored bars indicate the concentrations of each cytokine in the culture.

This cell line has a large output of IFNγ as well as some IL-10 and IL-13. While it appears that there was also an increase in IL-8 production, keep in mind that there was 1,000 pg of IL-8 to start with, so the increase is not as substantial as it appears.

You may be surprised that some cytokines are absent from the culture, specifically IL-2, but remember that IL-2 tends to be consumed about as fast as it is produced, so it’s usually not detectable.

Figure 1

Figure 2 shows similar data, but here we are stimulating PBMC instead of antigen-specific T cells. We analyzed the kinetics of cytokine production over the first three days in culture using CMV antigen and tetanus toxoid as stimuli. As with Figure 1, we again see IFNγ produced in abundance. We detected some IL-2 from these PBMC, although IL-2 was more present in the sample cultured with tetanus toxoid.

Figure 2

Intracellular Staining

Intracellular cytokine staining allows you to identify the cell population that’s involved in the response and quantitate the reactive cells.

Here is our method for intracellular cytokine staining:

1. Incubate antigen-presenting cells with peptide overnight
2. Add T cells
3. Continue co-culture overnight
4. Add brefeldin A for 4 hours
5. Stain dead cells and CD8
6. Fix and stain for IFNγ
7. Permeabilize the cells with 0.5% saponin

The addition of brefeldin A in the final four hours of culture prevents secretion of the cytokines. We also finish by permeabilizing the cells so the antibody can gain access to the inside of the cells.

Figure 3 shows the results of intracellular staining. The red histograms represent co-cultures with irrelevant peptide included as a negative control to set the baseline for staining, and the black histograms include the peptide recognized by the T cells.

Using CD8, we were able to analyze the antigen-specific T cells to show that the fluorescence shifted to the right when stained with IFNγ, indicating brighter fluorescence compared to the control. When focusing on the antigen-presenting cells in the culture, the histograms showing the background fluorescence and fluorescence in the presence of the relevant peptide are completely overlapping, indicating that there was no specific IFNγ signal in those cells and demonstrating that the CD8+ cells are in fact the cells producing IFNγ in the culture.

Figure 3

Ready to Learn More?

If you have specific research questions, you can always get a quick answer by asking a scientist, or by submitting a custom research request for additional help in your lab.