Optimizing Immunophenotyping Assays

Immunophenotyping assays use fluorophore-labeled antibodies to characterize cellular populations by flow cytometry. Until relatively recently, it was common practice for researchers to measure 4–6 markers in parallel. However, with the development of novel reagents and advanced flow cytometry instrumentation, more than 20 markers can now be measured simultaneously. As panel size increases, experimental design inevitably becomes more complex. This article offers guidance for optimizing immunophenotyping assays and includes tips for constructing multicolor flow cytometry panels.

Proper experimental design and planning is critical

Immunophenotyping assays present several unique challenges for experimental design. Critically, fluorophores must be compatible with one another to avoid generating misleading results. Equally important is the need to match antigen density to fluorophore brightness; scarce antigens should be paired with bright fluorophores, and vice versa, to ensure accurate detection. Where both extracellular and intracellular markers will be measured, it can often be necessary to stain for cell surface markers before fixation so that antibody binding epitopes are not affected by the fixative agent; for intracellular staining, permeabilization is essential. More general considerations include selecting the right sample type, titrating antibody reagents, and adjusting the flow cytometer settings using proper controls.

Know your fluorophores and instrumentation

In addition to fluorophore brightness, the excitation and emission maxima of each fluorophore must also be considered during panel design. These values define fluorophore compatibility with the flow cytometer’s lasers and detectors, respectively, as well as determine which fluorophores can be used together. Where two fluorophores share similar excitation maxima but have a different Stokes shift (the difference between the maximum excitation and emission wavelengths), they can often be combined in the same experiment.



In situations where the Stokes shift is comparable, replacing one fluorophore with a tandem dye is a popular approach to increase panel size; for example, both PE and the tandem dye PE/Cyanine7 share the same excitation wavelength, but PE/Cyanine7 has a much higher Stokes shift, altering its emission wavelengths to near 780 nm. For longer duration flow cytometry studies, or those using strong lasers, fluorophores that are resistant to photobleaching are recommended. Fluorophore properties and instrument specifications must always be assessed in parallel to understand the technical capabilities and limitations of the system.


Research the underlying biology

Understanding the biology of the various markers used for immunophenotyping is crucial. Factors to consider include whether healthy samples exhibit higher expression of certain markers compared to diseased samples; if some form of treatment (e.g., stimulation with a growth factor or dosing with a drug) is necessary for markers to be expressed at detectable levels; and whether cell surface markers are internalized under specific conditions. Sample preparation can also influence results, with enzymatic digestion of tissue material having the potential to impact surface marker integrity, and methods for lysing red blood cells (to prevent them from sequestering antibodies and other reagents) being associated with leukocyte loss where not performed correctly. Ensuring the right samples are used, and that they are handled appropriately, is key to producing reliable data.

Importance of experimental controls

Controls are used for monitoring assay performance and validating results. But while any well-designed assay will include biological controls (samples known to express, or lack, the target of interest), immunophenotyping assays require several additional controls particular to flow cytometry. Included among these, unstained controls determine the level of background fluorescence and are used for setting negative gates, whereas isotype controls (antibodies sharing the same isotype as a target-specific antibody but that do not recognize the target) are used to assess non-specific antibody binding (e.g., to Fc receptors). When building multicolor flow cytometry panels, fluorescence minus one (FMO) and compensation controls are critical. FMO controls comprise samples stained with all but one of the fluorophores and are used to evaluate fluorescence spread. In contrast, compensation controls are samples stained with just a single fluorophore and enable correction for spectral overlap.

Leverage open-access resources to streamline experimental design

A broad selection of resources is available to researchers wishing to perform immunophenotyping assays. Sites such as PAXdb, UniProt, and the Human Protein Atlas provide information about protein abundance, known isomers, and post-translational modifications, while antibody datasheets include validation data and recommended protocols that provide a good starting point for performing in-house research. Other useful tools include Spectra Analyzers for comparing laser lines, custom filters, and the excitation and emission profiles of fluorophores to establish a best fit, and Panel Builder tools to simplify the process of panel design. In addition, Optimized Multicolor Immunofluorescence Panel (OMIPs) are peer-reviewed panels detailing sets of validated reagents for characterizing a specific cell type or response.


BioLegend offers a broad selection of reagents and tools for optimizing immunophenotyping assays, including flow cytometry tools and fluorophore families.


This article originally appeared on Biocompare.


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