How To Do Phospho-Flow Cytometry

I often have researchers come into the core wanting to look at the activation and downstream signaling events that occur in different immune cells.

These events occur in response to signals such as cytokines, chemokines, various receptor ligands, and the engagement of the T cell or B cell receptors. The signaling events are also characterized by the initiation of several phosphorylation events.

Measuring Phosphorylation Events

When this is the case, I recommend that the researchers set up a phospho-specific flow cytometry, or phospho-flow, experiment. These types of experiments measure the phosphorylation state of intracellular proteins at the single cell level.

Phospho-flow allows for the analysis of many phosphorylation events, along with cell surface markers, simultaneously. These types of experiments enable the experimenter to resolve complex biochemical signaling networks in heterogeneous cell populations. Phospho-flow has been applied to numerous areas of biology, including antigenic stimulation and microbial challenge, high-throughput and high-content drug discovery, as well as the characterization of signaling pathways in both normal and disease-altered immune responses.

Fixing Your Cells For Phospho-Flow

In phospho-flow, immune cells or other cell populations are stimulated with signaling receptor ligands or antagonists for a certain period of time. Following stimulation, the cells are fixed using paraformaldehyde-based buffers. The fixation process locks the cells in their induced states of phosphorylation so they can be permeabilized and stained with fluorescently-labeled antibodies against the phosphorylated proteins.

The cells can also be stained with antibodies against cell surface markers and other proteins of interest at the same time. The final step is to analyzed the stained populations with a flow cytometer or cell sorter.

5 Phospho-Flow Tips

When performing and optimizing a phosphor-flow experiment, there are several things to consider.  Here are 5 ways to optimize your phospho-flow experiment.

1. Run all of your samples at the same time. 

When analyzing phoso-flow data, there are two things to keep in mind. First, the fluorescence intensities of each population will serve as a measure of the magnitude of the protein target’s phosphorylation levels. Second, the staining intensities will allow you to calculate the percentage of cells able to respond to a given signal.

The problem is that very often you will need to stimulate different cell populations on different days and at different times. This will create variability between staining intensities.

The best way to limit this variability is to use a buffer that allows you to freeze your all of your populations before adding antibody. Then, an hour or two before you’re ready to run your samples on a flow cytometer, thaw all of your samples at once. Then simply stain them and run them as one large batch.

2. Select the right permeabilization method.

Many protocols use 100% methanol for permeabilization following fixation. The advantage of this is that following methanol permeabilization, the cells can be stored for an extended period of time at -20°C to -80°C prior to staining.

This means you can stimulate different samples on different days and then run them altogether in one big batch to get more accurate results. All you have to do is keep freezing your cells after every stimulation.

3. Select the right target antigens.

Not all protein targets are created equal. Some antigens will not survive the permeabilization process, even if you’re using methanol. The best way to determine which antigens you should use is to review the literature and see which proteins remain stable during permeabilization.

You can also use online resources such as Fluorish and Cytobank to identify potential protein targets on your cell populations on interest.

4. Make sure you’re targeting the right event.

To measure phosphorylation events uniquely, you have to use antibodies that are specific to the phosphorylated form of a protein. These antibodies are usually raised using short phosphorylated peptide immunogens that are coupled to carrier proteins.

However, sometimes you’ll want to use several antibodies against the same phosphorylated protein. The key is that each antibody will target a different phospho-residue within the protein. The advantage of this is that you can gain insight into which residues are important for particular signaling events.

5. Make sure you’re ONLY targeting the right event. 

Once you’ve ensured that your antibody is targeting the right phospho-residue, you’ll want to confirm that your antibody is ONLY targeting that residue. In other words, you’ll want to confirm phosphor-specificity. There are several ways to confirm this.

First, you can compare the staining intensities of resting versus stimulated cell populations. Second, you can treat your samples with phosphatases prior to flow analysis. Third, you can compare phosphorylated peptides to non-phosphorylated peptides. Fourth and finally, you can compare phospho-protein levels to total protein content.

Additional References
Krutzik et al. Journal of Immunology, 2005, 175:2357-2365
https://www.bdbiosciences.com/research/ics/
http://www.cytobank.org/nolanlab/experiment_protocols/

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Tim Bushnell, PhD
Tim Bushnell, PhD

Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.

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