3 Must-Have High-Dimensional Flow Cytometry Controls

Developments such as the recent upgrade to the Cytobank analysis platform and the creation of new packages such as Immunocluster are reducing the computational expertise needed to work with high-dimensional flow cytometry datasets. Whether you are a researcher in academia, industry, or government, you may want to take advantage of the reduced barrier to entry to apply high-dimensional flow cytometry in your work. However, you’ll need the right experimental design to access the new transformative insights available through these approaches and avoid wasting the considerable time and money required for performing them. As with all experiments, a good design begins with the right controls. Here are the ones you need if you’re performing high-dimensional flow cytometry.

1. Batch controls

All experiments must control for variability in protocol performance among preparations or batches. Batch controls (or reference controls) serve this purpose in flow cytometry. Such controls allow you to catch pipetting errors, changes in instrument calibration, and more. They can also provide a means for potentially correcting for batch effects during analysis. Last but not least, they can be used to train others on your protocol. 

You should take time to identify an appropriate batch control before you even begin validating your experimental design. This control will be stained every time you stain an experimental sample. As such, it should be readily accessible and available in large quantities; you want to have more than enough for every preparation you may possibly wish to perform. It should also reflect the expected staining pattern in sufficient detail to allow you to confirm that your targets have been properly labeled. A large number of frozen peripheral blood mononuclear cells (PMBCs) from a single source, commercially available control cells, and beads are examples of good choices depending on your specific application. 

As an aside, additional steps should be taken to limit the variation between batches in high-dimensional flow cytometry experiments. This includes barcoding, the labeling of individual samples with a unique barcode, and sample pooling prior to antibody staining. The preparation of a large aliquot of stable sample staining master mix that can be used for every batch you process as part of your experiment is another step you can take.

2. Reagent controls

Reagent controls are used for the validation of flow cytometry experimental design. There are two types of reagent controls: titration controls and isoclonal controls.

Titration controls allow you to validate the amount of antibody used for staining. This validation should be accomplished via a titration experiment, and you will need to do such an experiment for each of the 30-50 antibodies you want to include in your high-dimensional flow-cytometry panel. To perform your titration experiments, vary the amount of antibody used in staining, while holding other variables such as incubation time, temperature, and cell concentration constant. Acquire data using an appropriate instrument and calculate the staining index (SI) for each concentration to create a plot similar to the one that appears in Figure 1. You will use this plot to identify the antibody concentration that is in the middle of the range that provides the highest SI. In this way, you ensure optimal sensitivity in your experiments.

Figure 1: Example staining index (SI) vs. antibody concentration plot obtained via a titration experiment.

Isoclonal controls are used to confirm that the cells are binding specifically to the antibody for the marker of interest rather than the label attached to the antibody. This confirmation is achieved through competition experiments. In these experiments, an unlabeled antibody of the same clone is added to samples in increasing concentrations to compete with the binding of the original antibody. If binding is specific, increasing the ratio of unlabeled antibody will result in a decrease in staining as shown in Figure 2.

Figure 2: Example of the results expected in an isoclonal control experiment if antibody binding is specific.

3. Gating controls

As in all flow cytometry experiments, high-dimensional flow cytometry experiments require a gating strategy that defines what is and what isn’t part of a particular cell population of interest. Otherwise, researchers open themselves up to the possibility of wasting time with irrelevant or biologically impossible results. Gating controls help researchers avoid this outcome. Which gating controls are best for your high-dimensional flow cytometry experiment depends on whether you are using polychromatic flow cytometry or mass cytometry.

There are 3 major types of gating controls for polychromatic flow cytometry: internal negative controls (INC), unstimulated controls, and fluorescence minus one (FMO) controls. While unstained samples are also sometimes used as negative gating controls, the truth is that these cannot properly define background.

Internal negative controls (INC) are cells in the staining sample that do not express the marker of interest. They take advantage of the biology that is known about the system under investigation. Assuming that such controls can be confirmed in the literature and through experimentation, they provide a robust control for proper gate placement.  

Unstimulated controls are samples that have not received a treatment used to activate a biological system. As such, they should not express markers associated with activation. Like INCs, this type of gating control can be very helpful for setting the proper gate because it takes the background binding of the target antibody into account.

FMO controls are samples that are stained with all except one of the fluorochromes used in a panel. These powerful controls reveal the spread of the data and are the best way to deal with the crowded spectra created by all the colors used in polychromatic flow cytometry. There will be one FMO control for every fluorescently labeled antibody used in the experiment. All of these should be run during the panel development phase. Afterward, only those controls that have been proven essential for identifying the target cells need to be run.

In mass cytometry, gating controls are different because there is no autofluorescence. Background in these experiments can come from overlap between mass channels or isotope impurity instead. The best way to avoid overlap and isotope impurity is through careful planning in the panel design and sample preparation phase. Mass Minus One (MMO) controls, which are similar to FMO controls, may also be used to resolve signal from background. 

Batch, reagent, and gating controls are all important for ensuring reproducibility and proper data interpretation in high-dimensional flow cytometry. To make the most of the increased accessibility of these experiments, make sure you choose the right controls for your particular study before diving in. Doing so will help you make sure you can get your results past a grant or manuscript reviewer so that any new transformative insights you uncover can be readily shared to benefit science overall.

To learn more about important control measures for your flow cytometry lab, 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.

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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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