When To Use (And Not Use) Flow Cytometry Isotype Controls

Antibodies can bind to cells in a specific manner – where the FAB portion of the antibody binds to a high-affinity specific target or the FC portion of the antibody binds to the FcR on the surface of some cells.

They can also bind to cells in a nonspecific manner, where the FAB portion binds to a low affinity, non-specific target. Further, as cells die, and the membrane integrity is compromised, antibodies can non-specifically bind to intracellular targets.

The question has always been how to identify and control for the nonspecific antibody binding observed.  

This led to many research groups using a control called the Isotype control.

The concept of this control is that an antibody targeting a protein not on the surface of the target cells, has the same isotype (both heavy and light chain) as the antibody of interest.  When used to label cells, those that showed binding to the isotype, would be excluded as they represented the non-specific binding of the cells.

When Isotype Controls Were Everything

Isotype controls were once THE negative control for flow cytometry experiments.

They are still very often included by some labs, almost abandoned by others, and a subject of confusion for many beginners. What are they, why and when do I need them? Are they of any use at all, or just a waste of money?

Most importantly, why do reviewers keep asking for them when they review papers containing flow data?

Isotype controls were classically meant to show what level of nonspecific binding you might have in your experiment. The idea is that there are several ways that an antibody might react in undesirable ways with the surface of the cell.

Not all of these can be directly addressed by this control (such as cross-reactivity to a similar epitope on a different antigen, or even to a different epitope on the same antigen). What it does do is give you an estimate of non-specific (non-epitope-driven) binding. This can be Fc mediated binding, or completely nonspecific “sticky” cell adhesion.

In order to be useful, the isotype control should ideally be the same isotype, both in terms of species, heavy chain (IgA, IgG, IgD, IgE, or IgM) and light chain (kappa or lambda) class, the same fluorochrome (PE, APC, etc.), and have the same F:P ratio. F:P is a measurement of how many fluorescent molecules are present on each antibody.

This, unfortunately, makes the manufacture of ideal isotype controls highly impractical. 

There is even a case to be made that differences in the amino acid sequence of the variable regions of both the light and heavy chains might result in variable levels of undesirable adherence in isotypes versus your antibody of interest.

Moving Beyond Isotype Controls

For these reasons, many in the field are moving beyond the isotype control. With some suggesting they be left out of almost all experiments.

If you spend any time browsing the Purdue Cytometry list, you’ll see these same arguments presented in threads about isotype controls. 

The following paper presents options for controls in several categories, the options available, and pros and cons of each option. The section on isotype controls summarizes the problems with the use of isotype controls very clearly…

Flow cytometry controls, instrument setup, and the determination of positivity.

Additionally, the following paper presents options for controls in several categories, the options available, and pros and cons of each option…

Considerations for the control of background fluorescence in clinical flow cytometry.

The section of the above paper focusing on isotype controls summarizes the problems with their use very clearly.

The article also illustrates difference in undesirable binding at different levels using the same clone from different manufacturers.

See the figure below for an example of how even the same isotype control clone can result in highly variable levels of undesirable staining.

Isotype Antibody | Expert Cytometry | Fluoresence

If you do use isotype controls in your experiment, they must match as many of the following characteristics as possible for your specific antibody—species, isotype, fluorochrome, F:P ratio, and concentration.

9 Tips For Using (Or Not Using) Isotype Controls

1. You certainly don’t need them for things that are clearly bimodal. If you are looking for T cells and B cells in peripheral blood the negative cells also in the circulation provide gating confidence. As seen in the example below, it is extremely easy to pick out CD4 and CD8 positive cells in the sample of lysed mouse blood.

Isotype Matched Control | Expert Cytometry | igg Isotype


2. If you are using post-cultured cells, the isotype control might give you some information about the inherent “stickiness” of your cells. However, this is not meant as a value you can subtract from your specific antibody sample in terms of fluorescence intensity or percent positive. This is simply a qualitative measure of “stickiness” and the effectiveness of blocking in your protocol.

3. If you are using multiple dyes in your search, and your concern is positivity by spectral overlap, you will be better served by using a fluorescence-minus-one control (FMO), in which all antibodies are included except the one you suspect is most prone to error from spectral overlap.

4. You should absolutely not be using them to determine positive versus negative cells, or as a gating control in your assay.

5. Keep in mind that the best way to avoid high levels of background staining of antigen-negative populations is to carefully titrate your reagents to ensure the highest positive signals in bright populations, while reducing spread in the negative population.

6. If you are using your isotype control and you are seeing high levels of non-desired staining, it is time to look carefully at your blocking step in your protocol. Are you using an Fc-block if you have myeloid cells? Have you tried adding excess immunoglobulins or whole serum to your buffer? Each of these can help pull down your nonspecific adherence.

7. Are you certain it is non-specific antibody adherence you are are dealing with and not free fluorochrome adherence? You can find out by using an isoclonic control. If you add massive amounts of non-fluorochrome conjugated monoclonal antibody to your staining reaction, your fluorescence should drop. If it does not, your issue is not due to nonspecific antibody binding, but to free fluorochrome binding.

8. For cell signalling and cytokine staining, besides the FMO control, make sure not to neglect a biological negative control, whether that be unstimulated cells, or cells treated with an inhibitor of phosphorylation.

9. Don’t forget to use a viability dye in the polychromatic panel. With the proliferation of these dyes in different colors and for both viable and fixed cells, there is no reason to not use these dyes. Viability dyes are essential for removing dead cells that will non-specifically uptake antibodies.

In conclusion, isotype controls are useful for demonstrating that there was poor blocking of the cells. They should never be used to determine or set positivity in fully stained samples.

To learn more about which controls you should use for your flow cytometry experiments 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

What Is Total Internal Reflection Fluorescence (TIRF) Microscopy & Is It Right For You?

What Is Total Internal Reflection Fluorescence (TIRF) Microscopy & Is It Right For You?

By: Heather Brown-Harding, PhD

TIRF is not as common as other microscopy based techniques due to certain restrictions. We will discuss these restrictions, then analyze why it might be perfect for your experiment.  TIRF relies on an evanescent wave, created through a critical angle of coherent light (i.e. laser) that reaches a refractive index mismatch.  What does it mean in practice?  A high angle laser reflects off the interface of the coverslip and the sample. Although the depth that this wave penetrates is dependent on the wavelength of the light, in practice it is approximately 50-300nm from the coverslip. Therefore, the cell membrane is…

5 Drool Worthy Imaging Advances Of 2020

5 Drool Worthy Imaging Advances Of 2020

By: Heather Brown-Harding, PhD

2020 was a difficult year for many, with their own research being interrupted- either by lab shutdowns or recruitment into the race against COVID-19. Despite the challenges, scientists have continued to be creative and have pushed the boundaries of what is possible. These are the techniques and technologies that every microscopist was envious of in 2020. Spatially Resolved Transcriptomics Nature Methods declared that spatially resolved transcriptomics was the 2020 method of the year. These are a  group of methods that combine gene expression with their physical location. Single-cell RNA sequencing (scRNAseq) was originally developed for cells that had been dissociated…

Picking The Right Functional Imaging Probe

Picking The Right Functional Imaging Probe

By: Heather Brown-Harding, PhD

As biologists, we study the process of life, however, it’s intricacies cannot be captured by a snapshot in time. Generally, the easiest imaging experiments are those where the samples are stained, fixed, and imaged within a few days of procurement, but that too doesn’t capture the dynamic processes common in cells and organisms. Live cell imaging when combined with reporters serves as a powerful tool to provide solid imaging data. Cameleon —one of the first reporters— was developed in 1997 in Roger Tsien’s lab.  Cameleon is a green fluorescent protein (GFP) that undergoes a conformational change in the presence of…

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.

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.