Intracellular Cytokine Staining with Flow Cytometry

Intracellular cytokine staining (ICS) with flow cytometry is a popular method for studying cellular responses under various experimental conditions. This article shares best practices for performing these types of experiments.

Advantages of ICS with flow cytometry

Cytokines are soluble proteins, secreted from a variety of cells, with important roles in mediating immune and inflammatory responses. Techniques used for their detection include ELISA, a bulk population method that usually measures just a single cytokine; ELISpot, which provides single-cell information at the expense of throughput; and RT-qPCR, which can identify the level of cytokine transcription in individual cells only when combined with a complex sample-prep workflow. An alternative approach, ICS with flow cytometry, overcomes the limitations of these methods.

“A major advantage of ICS with flow cytometry is that it offers an in-depth characterization of cellular functionalities,” says Maria Jaimes, Vice President, Scientific Commercialization, Cytek Biosciences. “This is possible for several reasons. First, multiple cytokines can be detected simultaneously on a single-cell basis. Being able to determine co-expression is important because cytokine production is not restricted to specific cell subsets, as was previously thought, but instead depends on environmental cues such as specific tissues or pathological conditions. Second, the phenotype of the cytokine-producing cells can be assessed based on the expression of cellular markers. Moreover, ICS can be combined with other functional assays such as proliferation, transcription factor expression, degranulation, and activation, thus providing a detailed readout of each cell’s functional status.”

“A further advantage of ICS with flow cytometry is its high throughput, which allows for analyzing thousands of cells per second,” adds Amber Miller, Ph.D., Flow Cytometry Scientist and IHC Supervisor at Bethyl Laboratories (a Fortis Life Sciences Brand). “Compared with other techniques used for cytokine analysis, ICS with flow cytometry provides more data per sample.”

The experimental workflow

A typical workflow for ICS with flow cytometry begins with cell activation. “Unless you are looking at cells that are already activated, such as in some ex vivo studies of peripheral blood mononuclear cells (PBMCs) in chronic infections, a form of activation is required,” explains Laurissa Ouaguia, Ph.D., Scientist, Reagents and Application Development, at Agilent Technologies. “Critically, this should induce the same cytokine profile as the physiological stimulus.”

At the same time, or immediately after activation, one or more protein transport inhibitors is added to block cytokine vesicle secretion and promote intracellular cytokine accumulation. “Brefeldin A and monensin are the most common cytokine secretion inhibitors,” reports Miller. “They can often be used interchangeably, although this is not always the case. For example, brefeldin A has been shown to trap more TNF-α inside activated cells than monensin.”

Next, the cells are harvested and stained to assess cell viability, prior to antibody-based detection of surface markers. “Some surface markers are downregulated due to membrane recycling, the extent of which depends on the stimulation method used,” cautions Jaimes. “For example, stimulating T cells with PMA and ionomycin leads to significant downregulation of CD4. Therefore, it is recommended that you investigate whether the surface markers you will use are affected by the stimulation conditions.”

Once surface marker staining is complete, the cells are fixed and permeabilized. “Fixation and permeabilization allow antibodies to access intracellular proteins,” says Miller. “But because these steps can adversely affect antibody staining and fluorophore intensity, optimization is required to assess whether they should be performed before or after staining for surface markers. This should include determining the optimal antibody titration, which can be different for live and fixed/permeabilized cells.”

Intracellular cytokine staining is then performed, followed by an (optional) fixation step to stabilize the signal, and the data is acquired on a flow cytometer. Here, Ouaguia recommends acquiring a large number of total events to accurately evaluate cytokines expressed at low frequencies. “You should also perform ‘backgating’ to assess the side scatter/forward scatter (SSC/FSC) characteristics of the cells gated on the cytokine fluorescent parameters,” she says.

Fixation and permeabilization challenges

Besides the potential to compromise antibody staining and fluorophore intensity, fixation and permeabilization can present further challenges. “Both of these steps can increase cellular autofluorescence,” comments Ouaguia. “In addition, permeabilization can increase the amount of protein available for nonspecific binding. These effects should be investigated using suitable controls during assay development, when optimization can help to minimize their impact.”

Ways of addressing residual autofluorescence include using bright fluorophores that emit outside of the green region (where most autofluorescence is seen) for detecting low abundance targets, and acquiring data on a spectral cytometer if available. “With spectral cytometers, cellular autofluorescence can be characterized and extracted,” explains Jaimes. “This results in better resolution and identification of the populations detected with fluorophores that emit at the same wavelengths as sample autofluorescence.”

Another problem linked to fixation and permeabilization is that these steps decrease cell size. “This can result in cells not being ‘on-scale’ with the scatter gains used for similar non-fixed/non-permeabilized cells and will require optimizing the cytometer scatter gains,” says Jaimes. “You may also need to adjust the speed of any centrifugation steps in order to prevent cell loss.”

Tips for intracellular cytokine staining with flow cytometry

The following suggestions are based on the combined expertise of all three contributors to this article:

• When optimizing the activation conditions, check that these induce the same cytokine profile as the physiological stimulus
• Ensure you are using the right cytokine secretion inhibitor for your model system
• Confirm that your cell viability stain is compatible with fixation and permeabilization
• Check that the surface markers you intend to use are not affected by the stimulation conditions
• Be extra cautious when optimizing the fixation and permeabilization steps
• Always include appropriate controls—e.g., stimulated and unstimulated samples to establish basal expression levels, unstained controls for identifying autofluorescence, single staining controls to detect non-specific antibody binding
• Optimize the number and duration of any wash steps to ensure unbound antibodies are removed
• Select fluorophores with care—pair brighter fluorophores with less abundant targets and remember to consider autofluorescence
• Acquire a large number of total events and remember to perform ‘backgating’ to assess the SSC/FSC characteristics of the cells gated on the cytokine fluorescent parameters
• If you are unsure about whether your cytokine staining is real, check to see if staining is reduced/eliminated by the addition of recombinant cytokine