Why You Need To Use FMO Controls For All Multicolor Flow Cytometry Experiments

What are the three most important parts of any flow cytometry experiment?

Controls, controls, controls.

Of course, this is mostly in jest. Mostly.

Reagent selection, well-maintained instruments, and other factors are also important. However, you must control for these factors.

Setting up the right controls is critical to determining how your cells are responding to treatment. It’s also important for correctly interpreting your data and drawing correct conclusions. Without the proper controls, you would not be able to compensate your flow cytometry experiments correctly or identify your cells of interest correctly.

By using the wrong controls, or leaving out certain controls altogether, you leave yourself wide open for criticism. Your gate placement will be questioned, your samples will be questioned, and your papers and grants will be questioned.

Why Fluorescence Minus One, Or FMO Controls, Are Important

We’ve all had experience working with someone who wanted to see the “real” samples, not the control samples, first.

But if you’re a smart scientist, you know “real,” or experimental samples are meaningless unless you know, among other things, the background levels you’re working with.

Valuing your control samples over your experimental samples is a mistake, especially when performing flow cytometry experiments. For example, in flow cytometry, some gates are “easy” to set. CD19+ B cells are often easy to pick out on a plot showing CD3 for a T cell subset sample. Other gates, however, are more difficult to define. This is especially true when you’re looking at activation markers within a continuum, or dim levels of positivity, like FoxP3 in regulatory T cells. How can you convince reviewers that you didn’t make an error and placed your gate in the proper place?

Now, consider gating out subsets in complicated 10+ color experiments. How are you going to account for the data spread that occurs with compensation? In any multicolor flow cytometry experiment, the answer to your gating troubles is to use Fluorescence Minus One, or FMO, controls.

What Is A Fluorescence Minus One, Or FMO, Control?

FMO controls are samples that contain all the antibodies you are testing in your experimental samples, minus one of them.

When analyzing the minus, or left out parameter in an FMO control, you give yourself a strong negative control to work with. It’s a strong negative control because the left out marker in the FMO control allows you to take into account how the other stains in your panel affect the left out parameter.

FMO controls are required for accurately discriminating positive versus negative signals, high versus low (or variable) antigen expression levels, and more. Even simple 2- or 3-color experiments reveal the need for FMO controls when drawing gates.

The figure below shows data from an experiment that was set up to identify naive versus memory CD4+ T cells. After properly compensating the data, the experimenter is left with the plot on the far right. Now the question becomes, where does the experimenter set the gate for PE positivity?

If the experimenter uses an unstained control only (far left panel), he or she is able to set a boundary (red dashed line) for PE positivity. However, if you now look at the middle plot, you can see that the bulk of the cells are labeled as “positive” for PE because they are above the boundary established by the unstained control.

multicolor flow cytometry training | Expert Cytometry | fmo control flow cytometry

Notice that the cells in the middle plot are stained with all the fluorochromes in the experimental samples except PE. As a result, they can NOT be positive for PE. This middle panel represents the FMO control in this experiment and shows the experimenter that the true negative boundary for PE, or FMO bound, is where the blue line lies.

Now, using the FMO control and resulting FMO bound, the experimenter is able to set the proper gate and ensure that the spread of the data (blue double arrow on the far right), is not impacting his or her gating.

In the more complex example below, the plots show a population of circulating CD34+ cells in human PBMCs. The experimenter who ran these samples wanted to determine the levels of CD146 on these cells. The experimenter included FMO controls when testing the panel (CD146 on PE and CD31 on FITC, bottom two plots). As a result, the experimenter was easily able to draw gates around the populations of interest despite low levels of background positivity (top two plots).

multicolor flow cytometry training | Expert Cytometry | fmo control flow cytometry

When To Use Fluorescence Minus One, Or FMO Controls

  1. When running any multicolor flow cytometry experiment, there is a spread in the data that quickly becomes apparent when you compensate your data. Due to the physics of fluorescence and the exponential scales used to display flow cytometry data, this spread is unavoidable. However, using FMO controls in your flow cytometry experiments can remove all ambiguity from your compensated multicolor plots.
  2. When antigens are dim or variably expressed, the use of FMO controls is critical. This is especially true when analyzing cell activation markers on immune cells. (Don’t forget those unstimulated biological controls here either!)
  3. Do NOT add isotype antibodies to your FMO controls. These antibodies do NOT add any useful information to the FMO. If you are working with myeloid cells and think you need an isotype control, prepare it separately.
  4. If you are unsure of the levels of expression you are dealing with in your samples or the sensitivity of your antibodies or samples, use FMO controls. It’s better to prepare a few controls you may not need than lose money, time, publications or grant funding by being stingy.

When in doubt, follow Maecker’s advice reported in Cytometry, “When high-quality monoclonal antibody conjugates are used at appropriate concentrations, they tend to have relatively low background staining. As such, in experiments of >4 colors, the major source of background staining tends to be fluorescence spillover. Because of this, the use of FMO controls has become both popular and prudent.”

Fluorescence Minus One, or FMO controls are critical for any scientist who wants to back up his or her drawing of flow cytometry gates. When you use FMO controls, you establish scientific evidence as to why the gates you drew are drawn correctly. This is true for any multicolor flow cytometry experiment. FMO controls will give you, as well as those who view your work with a critical eye, confidence in the degree of accuracy of your measurements.

To learn more about FMO controls, 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

6 Areas Of Consideration For Flow Cytometry Cell Cycle Analysis

6 Areas Of Consideration For Flow Cytometry Cell Cycle Analysis

By: Tim Bushnell, PhD

Cell cycle seems like such a straightforward assay. At its heart, it is a one-color assay and should be a simple protocol to follow. However, as discussed before, fixation and dye concentrations are critical. Once those are optimized, it becomes important to run the cells low and slow in order to get the best quality histograms for analysis — the topic of another blog. Adding the critical CEN and TEN controls will help standardize the assay, and ensure consistency and reproducibility between runs while helping identify non-standard (aneuploid, polyploid) populations from normal ploidy. Trying to isolate and focus on specific…

Why Cell Cycle Analysis Details Are Critical In Flow Cytometry

Why Cell Cycle Analysis Details Are Critical In Flow Cytometry

By: Tim Bushnell, PhD

Cell cycle analysis appears to be deceptively easy in concept, but details are absolutely critical. It is not possible to hide the data if there is poor sample preparation, incorrect dye ratios, too much (or too little) staining time, etc. Forgetting RNAse when using PI will doom your data to failure. Take these basics into account as you move into performing this simple, yet amazingly informative assay.

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…

5 Essential Beads For Flow Cytometry Experiments

5 Essential Beads For Flow Cytometry Experiments

By: Tim Bushnell, PhD

Flow cytometry is designed to measure physical and biochemical characteristics of cells and cell-like particles using fluorescence. Fundamentally, any single-particle suspension (within a defined size range) can pass through the flow cytometer. Beads, for better or worse, are a sine qua non for the flow cytometrist. From quality control,to standardization, to compensation, there is a bead for every job. They are important — critical, even — for flow cytometry.

How To Use Flow Cytometry To Measure Apoptosis, Necrosis, and Autophagy

How To Use Flow Cytometry To Measure Apoptosis, Necrosis, and Autophagy

By: Tim Bushnell, PhD

Using flow cytometry and a host of different reagents, it is possible to tease out how your cells may have died. Using these tools, you can readily eliminate the various suspects and come to your conclusion as to how your treatment may have killed your cells of interest. Here are some reagents to consider when measuring apoptosis, necrosis, and autophagy.

Flow Cytometry Protocols To Prevent Sample Clumping

Flow Cytometry Protocols To Prevent Sample Clumping

By: Tim Bushnell, PhD

Good flow cytometry depends on a high quality, single cell suspension. If the cells put through the instrument are not of high quality, the ensuing data will be difficult to analyze. Likewise, if the sample is clumpy, one will not be able to readily distinguish cells of interest from the clumps they are attached to. Sample preparation becomes the critical first step in any flow cytometry experiment. To get high quality results, follow these 3 sample preparation steps.

How To Compensate A 4-Color Flow Cytometry Experiment Correctly

How To Compensate A 4-Color Flow Cytometry Experiment Correctly

By: Tim Bushnell, PhD

Compensation in flow cytometry is a critical step to ensure accurate interpretation of data. It is also one of the areas that’s steeped in mystery, myths and misinformation. Manually adjusting the compensation values based on how the populations look, or so-called ‘Cowboy Compensation’, is not the correct way to determine proper compensation. The best practices for compensation involve following some very specific rules. Here are 4 steps to correctly compensating 4+ color flow cytometry experiments.

How To Differentiate T-Regulatory Cells (Tregs) By Flow Cytometry

How To Differentiate T-Regulatory Cells (Tregs) By Flow Cytometry

By: Tim Bushnell, PhD

T regulatory cells (Tregs), formerly known as T suppressor cells, are a T cell subset with direct roles in both autoimmunity and responses to pathogens. Tregs decrease inflammation via the secretion of immunosuppressive cytokines (IL-10, TGF-b) and also through direct suppression of inflammatory effector T cells (such as Th1 and Th17 cells). Given the importance of this unique T cell subset in so many immune responses, many investigators feel remiss if they immunophenotype their cell populations of interest without including a Treg measurement in the mix. But quantifying Tregs can be complicated. This article will show you how to quantify…

How Cell Culture Medium Can Decrease Cell Viability During A Flow Cytometry Cell Sorting Experiment

How Cell Culture Medium Can Decrease Cell Viability During A Flow Cytometry Cell Sorting Experiment

By: Tim Bushnell, PhD

When setting up a cell sorting experiment, there are many things to consider. You must consider which controls you’re going to use, how you’re going to compensate the experiment, which instrument and which instrument settings are ideal, and how you plan to analyze, gate, and present your data. With so many things to consider, it’s easy to lose site of the small things that can drastically affect the viability of your cells, including the composition of your suspension buffer. The composition of the suspension buffer for preparation, staining, analyzing and sorting is perhaps the most important parameter for maintaining viability…

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.