4 Considerations For Assessing Protein Phosphorylation Using Flow Cytometry

Signaling pathways are of great interest in many areas of research because dysregulation of these pathways can lead to diseases such as cancer and lupus. This also means these pathways contain potential therapeutic targets to help treat these diseases.

The power of flow cytometry is the ability to analyze millions of events in a short period of time. This allows for the analysis of dynamic cellular processes in a phenotypically defined manner.

The process of cellular signaling relies on changes in the phosphorylation state of proteins to either up or down regulate downstream processes (Figure 1).

Figure 1: The PI3Kinase signaling pathway has been implicated in cancer growth. From Vivance and Sawyer (2002) Nature Reviews Cancer 2:489-501

Initially, phosphorylation state analysis was performed by western blot — a bulk analysis method where subtleties of expression can be lost. To transition phosphorylation analysis to a flow cytometry assay, several factors needed to be optimized.

First, a source of high-quality antibodies to a very specific target needed to be produced. Second, the best way to stain the cells for specific, efficient labeling of targets while preserving the integrity of surface staining needed to be determined.

Proteins can be phosphorylated at 3 residues: Serine, Threonine, or Tyrosine. Phosphoprotein antibodies must be able to resolve a single change at the appropriate site in the presence of non-phosphorylated targets. When screening hybridomas, manufacturers must screen for both positive binding and absence of binding to the non-phosphorylated target.

The second concern was testing which protocols would be best for fixation and permeabilization of the cells to preserve the surface staining, the epitopes, and cell shape, while ensuring the antibodies were able to access the intracellular space.

If you are considering going after phosphorylation state, here are 4 important considerations that you should keep in mind when performing your experiments.

1. Choice of Instrument — while this may sound odd, there are 3 different classes of instrument on the market that can be used for cytometry-based assays. The traditional fluorescent flow cytometers, the spectral analyzers, and the mass cytometer. Each have their strengths when performing this assay.

  1. Traditional Fluorescent Flow — most readily accessible to the majority of users. There is a limited choice of fluorochromes for phosphoflow, which is impacted by the size of the fluorochrome and the autofluorescence of the cells. This limits the number of targets that can be measured in one assay.
  2. Spectral Analyzers — still relatively new to the marketplace. These systems should help improve resolution, as they are better able to handle autofluorescence of the cells. There are still limits because of fluorochrome size, but spectral analyzers can allow resolution of closely overlapping fluorochromes. As shown on this figure generated on the new Aurora from Cytek, they are able to resolve AF488 and AF532 in the presence of PE (Figure 2).

Figure 2: Emission resolution possible with spectral analyzers. Information from Cytek.

  1. Mass Cytometry — it is hard to beat the power and multiplexing capabilities of the CyTOF mass cytometer. Over 30 parameters are possible, and the use of isotope labeled antibodies alleviates size constraints. Likewise, there is no autofluorescence from the cells to reduce sensitivity. The high cost of adopting a mass cytometry panel, the limited availability of reagents, and potential data analysis complexity all reduce the attractiveness of this technology, but if you are seeking to screen a large number of targets, the mass cytometer is hard to beat.

2. Choice of Fluorochrome panel design for phosphoflow assay offers some additional challenges. First, is the consideration of the size of the fluorochrome.

After making holes in the membranes, it is important that the contents do not leak out, so the holes can’t be too large. We are trying to get antibodies into the cells, so imagine adding a PE molecule that is larger than a typical antibody and trying to get that into the cell.

That is why cyclic ring compounds like Fluorescein, Texas Red, and the Alexa type dyes are popular. These are relatively small (usually under 1000 g/mol), so don’t impact the size of the antibody as much as the larger fluorochromes do (Figure 3).

Figure 3: Size of some common fluorochromes including Fluorescein, Alexa 532 (carboxylic acid, succinimidyl ester shown), Phycoerythrin, allophycocyanin, and a typical antibody.

Once the fluorochrome choice is made, surface staining must be considered. This will be impacted in 2 ways.

First, the choice of phospho-antibody conjugate will remove some fluorochromes from consideration. Second, optimal fixation conditions for the phospho-antibody may negatively impact the surface stain fluor. Fortunately, there is a guide published by Cytobank to help make this decision.

3. Choice of Protocol — fixation and permeabilization protocols are critically important for success of these experiments. The same link from Cytobank can help with protocol choice. The choice of fixatives and the order that the fixatives are used is critically important to get right and optimize (Figure 4).

Figure 4: Effects of different fixation protocols on the surface staining of different clones and fluorochromes. From Perez et al., (2005) Curr Protoc Cytom. Unit 6.20.

4. Choice of Analysis — when done properly, this assay can generate a great deal of information quickly, and it is critical to find ways to present it both graphically and statistically. This can be done using heat-maps or other tools.

Figure 5: Heatmap analysis and clustering of 6 different phospho-Ab, either at basal or one of 5 different stimulation conditions. From Irish et al., (2004) Cell 118:217-228.

If you are interested, another way to look at this is a fold change. Jonathan Irish recommends using a transformed ratio, and in the simplest form it is:

log10(MFIstim) – log10 (MFIunstim)

Other data transformations exist, and these are discussed here.

Of course, the standard considerations for any flow cytometry experiment exist, such as appropriate samples, proper simulation, the necessary controls, and the protocols for setting up and running the instrument. Phosphoflow adds a level of complexity to this process, but it is not insurmountable.

If you are seriously considering adopting this assay into your research, it may be good to read and take the U937 challenge. This challenge is a perfect training tool to see how well you can perform and analyze phosphoflow data.

If you want more training, the Irish lab at Vanderbilt has put their training material on the web for everyone to use. You can also find some videos at the Nolan lab at Stanford.

For those working in the signaling field, having the ability to take a sample and phenotypically identify it, while knowing what is happening inside the cell to the target molecules of choice, opens up a host of new opportunities. These assays are amenable to high throughput setup, meaning that biologically relevant outcomes in pre-clinical drug discovery can be measured directly. All told, with a little forethought, some careful planning, and validation, phosphoflow assays are within your reach.

To learn more about the 4 Considerations For Assessing Protein Phosphorylation Using Flow Cytometry, and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.

Join Expert Cytometry's Mastery Class
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.

Similar Articles

We Tested 5 Major Flow Cytometry SPADE Programs for Speed - Here Are The Results

We Tested 5 Major Flow Cytometry SPADE Programs for Speed - Here Are The Results

By: Tim Bushnell, PhD

In the flow cytometry community, SPADE (Spanning-tree Progression Analysis of Density-normalized Events) is a favored algorithm for dealing with highly multidimensional or otherwise complex datasets. Like tSNE, SPADE extracts information across events in your data unsupervised and presents the result in a unique visual format. Given the growing popularity of this kind of algorithm for dealing with complex datasets, we decided to test the SPADE algorithm in 5 software packages, including Cytobank, FCS Express, FlowJo, R, and the original, free software made available by the author of SPADE. Which was the fastest?

5 FlowJo Hacks To Boost The Quality Of Your Flow Cytometry Analysis

5 FlowJo Hacks To Boost The Quality Of Your Flow Cytometry Analysis

By: Tim Bushnell, PhD

FlowJo is a powerful tool for performing and analyzing flow cytometry experiments, if you know how to use it to the fullest. This includes understanding embedding and using keywords, the FlowJo compensation wizard, spillover spreading matrix, FlowJo and R, and creating tables in FlowJo. Extending your use of FJ using these hacks will help organize your data, improve analysis and make your exported data easier to understand and explain to others. Take a few moments and explore all you can do with FJ beyond just gating populations.

Statistical Challenges Of Rare Event Measurements In Flow Cytometry

Statistical Challenges Of Rare Event Measurements In Flow Cytometry

By: Tim Bushnell, PhD

It is necessary to sort through hundreds of thousands or millions of cells to find the few events of interest. With such low event numbers, we move away from the comfortable domain of the Gaussian distribution and move into the realm of Poisson statistics. There are 3 points to consider to build confidence in the data that the events being counted are truly events of interest and not random events that just happen to fall into the gates of interest.

How to Optimize Flow Cytometry Hardware For Rare Event Analysis

How to Optimize Flow Cytometry Hardware For Rare Event Analysis

By: Tim Bushnell, PhD

Preparing for rare event analysis requires an understanding of the power and limitation of the instrument to be used. From how fast to run the fluidics, to how the signal is processed to the number of gates that can be used in the sorting experiment, each factor impacts the outcome of the experiment.

How To Choose The Correct Antibody For Accurate Flow Cytometry Results

How To Choose The Correct Antibody For Accurate Flow Cytometry Results

By: Tim Bushnell, PhD

With the added emphasis on reproducibility, it is critical to look at every step where experiments can be improved. No single step makes an experiment more reproducible, rather it is a process, making changes at each stage that leads to reproducibility. Antibodies comprise a critical component that needs to be reviewed. As Bradbury et al. in a commentary in Nature pointed out, the global spending on antibodies is about $1.6 billion a year, and it is estimated about half of that money is spent on “bad” antibodies. This does not include the additional costs of wasted time and effort by…

How To Achieve Accurate Flow Cytometry Calcium Flux Measurements

How To Achieve Accurate Flow Cytometry Calcium Flux Measurements

By: Tim Bushnell, PhD

Dyes exist for the detection of everything from large nucleic acids to reactive oxygen species, and from lipid aggregates to small ions. Concentrations of physiologically important ions such as sodium, potassium, and calcium can be important indicators of health and disease. Calcium ions play an especially critical role in cellular signaling. As a signaling messenger, calcium is involved in everything from muscle contractions, to cell motility, to enzyme activity. Calcium experiments can be very informative, and with the advent of cheaper UV lasers, more and more researchers can use ratiometric measurements to evaluate the signaling processes in phenotypically defined populations.

How to Perform Doublet Discrimination In Flow Cytometry

How to Perform Doublet Discrimination In Flow Cytometry

By: Tim Bushnell, PhD

You are probably familiar with the term, “doublet discrimination” or “doublet exclusion”, and have likely included this flow cytometry measurement into at least some (if not all) of your gating strategies. Even though you may utilize this important gating strategy, you may not have had the chance to delve deeper to explore exactly what doublets are and why it’s critical to exclude them. This article aims to give you insight on the what, why, and how of doublet discrimination.

5 Essential Calculations For Accurate Flow Cytometry Results

5 Essential Calculations For Accurate Flow Cytometry Results

By: Tim Bushnell, PhD

Flow cytometry is a numbers game. There are percentages of a population, fluorescence intensity measurements, sample averages, data normalization, and more. Many of these common calculations are useful, but surrounded by misconceptions. This primer will help you decide which calculation to use, when to use it, and how to interpret the results.

Measuring Receptor Occupancy With Flow Cytometry

Measuring Receptor Occupancy With Flow Cytometry

By: Tim Bushnell, PhD

Measuring the receptor occupancy of a given target showcases the power of flow cytometry. With the right reagents, best practices, and attention to detail, this assay can become a mainstay in your research toolkit. It extends quantitative flow cytometry to the next level, to determine a complete biological picture of how efficiently a given target is being bound. This also serves as the basis for even more fine-analysis when combined with assessment of downstream targets that the engagement of the receptor by the target antibody may affect. Phosphorylation, cell cycle arrest, and protein expression are all within reach, resulting in…

Top Technical Training eBooks

Get the Advanced Microscopy eBook

Get the Advanced Microscopy eBook

Heather Brown-Harding, PhD

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.

Get The Free Modern Flow Cytometry eBook

Get The Free Modern Flow Cytometry eBook

Tim Bushnell, PhD

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more.

Get The Free 4-10 Compensation eBook

Get The Free 4-10 Compensation eBook

Tim Bushnell, PhD

Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.