Antibodies 101: Flow Cytometry Controls

By Guest Blogger

Guest Blogger June 06, 2024

When you are running flow cytometry, you’ll need various controls to help you set up and analyze your samples. While you are probably familiar with the basics of controls in experimental design, you’ll need a few controls specific to flow as an application. These controls will allow you to distinguish real results from background noise or nonspecific binding.

Flow specific controls

The three types of flow cytometry controls we’ll discuss are single color, fluorescence minus one (FMO), and isotype. You should always have single color controls in your experiment, so we’ll go over those first. Depending on the markers you are looking at and the antibodies you are using, you may need to include additional controls like FMO or isotype controls, which we’ll describe a bit later.

Single Colors

Single color controls contain, as the name suggests, a single color from your antibody panel. They are made up of compensation beads and a conjugated antibody. Compensation beads are a mixture between synthetic beads that can bind to a conjugated antibody and beads that are unable to bind to them: like cells but with far more predictability. The beads create an artificial positive (bound) and negative (unbound) population. The benefit of beads is that they do not require cells from your experiment, and they are non-specific. The compensation beads can bind to any primary antibody you add.

Single color controls are used to help you set up your voltages and can also be helpful for compensation. You should have a single color control for every fluorophore you intend to use in your panel.

Using single color controls

On a flow cytometer, you generally want your positive cells to read between 10⁴ and 10⁵ in order to have enough room on the plot to see all of your positive and negative cells. This can be achieved by adjusting your voltage using your single color controls (Figure 1A), instead of using your actual samples. They may also be used as a starting point for compensation (Fig. 1B-C). However, it’s important to note that because you’re using beads and not experimental samples, the voltages and compensation you set from your single color controls may not be accurate for your samples. The single color controls should be used as a starting point, to preserve your sample, and you should then confirm or adjust the settings with experimental samples.

Three flow plots, labeled A, B, and C. A shows two clusters low on the y axis, in a straight line, one close to the X,Y intercept and one far from it. B shows a flow plot with one horizontal cluster low on the Y axis. Farther away on the X axis is a cluster in a diagonal line, pointing towards the upper right corner. C shows a flow plot with two clusters, one a horizontal line and further down on the X axis, a vertical line.

Figure 1: Example flow plots for a single color control. A) A flow plot showing a single color control for the APC channel. B) and C) A flow plot showing an APC single color control prior to compensation (B) and after compensation (C) with APC-Cy7.

FMOs

FMO stands for fluorescence minus one. It is used as a negative control for a specific marker in your experiment. To make an FMO sample, you take a subset of your experimental samples, combine it into one well or tube, and stain it with every marker you are using in the experiment except for one marker of interest. This allows you to more easily and accurately gate populations that may otherwise be difficult to separate out. In Figure 2, you can see that, without the FMO, it would be difficult to differentiate the Ly6C negative population from the Ly6C positive population.

Two flow plots labelled A and B. A shows a plot with a small, vertical cluster just outside an empty gated area. B shows the same population and gate, but now the population has a long horizontal tail that is present in the entirety of the gate.

Figure 2: An FMO gating example. The FMO sample (A) can be used while setting up your gating schematic to easily determine where your cells of interest are (B).

Using FMO controls

In an experiment, you should have a FMO sample for any marker without a clear positive and negative population. If you’re unsure what your population will look like for a particular marker, you can use the reference plots provided by the antibody companies or check the literature.

Pro tip! Early gates, like your live/dead markers or commonly used markers that define broader cell populations (e.g., CD4, CD8b, CD11b, CD19), are less likely to require an FMO. Markers that define more specific cell populations, often used in the later gates, are more likely to require FMOs.

Isotypes

Isotype controls are negative controls that allow you to determine what level of non-specific binding you have in your sample. The isotype antibody will be virtually identical to the antibody for your marker of interest. It will be from the same host species, have the same Ig subclass, and be in the same fluorophore (e.g., if you are staining for anti-mouse Ly6C in APC with the IgG2c subclass, you will use the APC IgG2c isotype control). The only difference is that the isotype antibody will not target your marker of interest.

Using isotype controls

An isotype sample is prepared very similarly to a FMO sample: you make a sample from a combined subset of your experimental samples, stain it with every marker you are using in the experiment except for your marker of interest, and then add in your isotype control for that particular marker. This enables you to identify any non-specific binding to know that your positive cells are truly positive for your marker of interest (Fig. 3).

Two flow plots, labelled A and B. A shows a single, gated population in a vertical cluster, close to the Y axis. B shows the same gated population, with a second, larger vertical population to the right of it. Both populations have tails going towards the other population.

Figure 3: Isotype flow plot example. The isotype sample (a) can be used to confirm that your positive population is specific for your target antibody and may assist in gating your target population (b).

There is some debate in the literature as to when you need isotype controls. Our lab prefers to use them to gate our populations, but others do not. Some journals require isotype controls, while others don’t. However, the general consensus seems to be that if you are looking for a rare population or are unsure what amount of antibody to use, you should consider using an isotype control.

Pro tip! Many companies will have appropriate isotype controls linked on their commercial antibody pages.

Flow cytometry can be a complicated and daunting process. Controls like single color controls, FMOs, and isotypes can help you more easily gate your target population. In addition, the controls will help you save on sample volume, so you can use most of your samples for flow analysis rather than for setting up your initial voltages and gates.

2023-12-11_091616950-min Ashlyn has a research background in CD8+ T cells and cancer immunotherapies. She is currently a research technician in Arlene Sharpe's lab at Harvard Medical School.