Flow Cytometry Strategies to Optimize Immunophenotyping

Immunophenotyping is one of the most common applications of flow cytometry, underpinning many different research programs. But while certain cell types can be identified by just a single marker, others require that multiple markers be used for characterization. This presents challenges for panel design, which are amplified by the fact that a typical immunophenotyping study involves detecting several cell types in parallel. In this article, we highlight some reasons for immunophenotyping cells and share flow cytometry strategies to help you succeed.

Immunophenotyping studies vary in complexity

“There are many reasons to immunophenotype cells,” explains Mike Blundell, product manager for flow cytometry reagents at Bio-Rad. “These include biomarker analysis in drug discovery and research; and disease diagnosis—which encompasses everything from identification of a malignancy or an immunodeficiency, to monitoring minimal residual disease (MRD) or relapse. Immunophenotyping is also essential for disease management and treatment planning, as well as providing insights into pathogenesis (e.g. the effect of the disease on the host immune system). Depending on the aim of the immunophenotyping study, the number of markers detected can vary considerably. Four markers would allow you to identify general lineages in the blood, for example, whereas for identification of specific lineage subsets this may rise to 8. Once you begin studying activation state, memory status, or subsets of multiple lineages, the number of markers detected simultaneously can rapidly rise to over 20.”

Larger panels offer higher rewards

Sarah Klein, Ph.D., senior development scientist at Cell Signaling Technology, comments that a major challenge of immunophenotyping is that the biology of certain immune cell markers is only now being investigated. “In many cases, researchers are still trying to understand the function of particular markers and how they lead to the immunophenotype they are trying to characterize,” she says. “For this reason, it is important to keep up to date as the literature matures. One tried and trusted approach to immunophenotyping is to start with a panel that includes basic markers for multiple immune cell types (e.g. CD3 for T cells, CD19 for B cells, CD14 for monocytes) then, once you have discovered which cell types vary most between your control and investigative samples, you can prepare panels targeting specific subtypes of immune cells (e.g. CD4⁺CD25⁺FoxP3⁺ Tregs and CD68⁺CD206⁺CD163⁺ immunosuppressive macrophages). Next, you can focus your studies on a smaller number of cell subtypes and investigate their function and signaling using some intracellular markers (e.g. CD8⁺Tox⁺TIGIT⁺PD-1⁺Tim3⁺CTLA-4⁺ exhausted T cells and CD4⁺Tbet⁺IFNγ⁺CD69⁺phospho-SLP76⁺ activated Th1 cells). These larger immunophenotyping panels are complicated to optimize and analyze, but offer a higher reward in trying to decipher the biology of immune cell types in the context of your study.”

Experimental considerations

Researchers have developed numerous strategies to optimize immunophenotyping studies. Dr. Alexandra Wittmann, senior scientist, flow cytometry, imaging team at Abcam, remarks that these include depleting red blood cells from whole blood or tissue isolated cell suspensions to simplify immune cell analysis, and including CD45 in staining panels as a pan-leukocyte marker to exclude non-immune cells during the gating process. “Another proven approach is to pair weakly expressed targets or very rare cell types with bright fluorophores, and vice versa,” she says. “Spectral overlap between dyes should also be considered since this will impact how much compensation is required; distributing dyes with overlapping spectra on different cells is preferential over binding to the same cells. Additionally, when establishing a staining panel, it is important to test various antibody concentrations to identify that which gives the best signal-to-noise ratio; it should never be assumed that experimental conditions will exactly match those tested by the antibody manufacturer.”

Stuart Williams, senior manager for scientific and application support at BD Biosciences, stresses that the most important aspect of any immunophenotyping study is knowing the biology of your system. “If you do not get that correct at the outset, all the assumptions made in your experiment will be wrong and your data quality will suffer as a result,” he reports. “There is often no one ‘right’ marker to identify a cell population; instead, several different markers may be required—and these may be either positive or negative. Whether you need to include or exclude events will depend on how your populations of interest are defined.” Lisa Bellemare, research applications scientist, also at BD, agrees, adding that selecting the right markers before choosing appropriate fluorophores is critical. “When it comes to marker selection, thoroughly analyzing the literature before starting to design the panel is key,” she says. “During this process, researchers should never overlook the value of antibody vendors' technical datasheets—these can really help with both creating and testing your hypothesis.”

The tips for success don’t stop here. Wittmann recommends fixing cells before intracellular staining to ensure the stability of soluble antigens or antigens with a short half-life, such that the target protein is retained in the original cellular location. She also notes that intracellular antigens require cell permeabilization before staining, while commenting that some permeabilizing agents (e.g. saponin) are reversible and must therefore be included in every wash step and antibody staining mix. Controls are of course vital to make sense of data; for flow cytometry, these should include unstained controls, isotype controls, and single-stained compensation controls, as well as fluorescence minus one (FMO) control samples. “You should also include a viability stain in your experiment,” mentions Bellemare. “Dead cells are great at binding antibodies, which can lead to unwanted false positive results.”

Lastly, Williams points out that immunophenotyping is incredibly powerful, making it worthwhile to take the time to get it right. “If you think of each marker as being a yes/no decision, then for every color you use, you’re doubling the number of cell types you can identify,” he says. “So, if you’re using an 18-color panel, you could potentially define over 262,000 cell populations—even more if you factor in negative/dim/bright expression of different markers. Recently, single-cell genomics has opened up new avenues to explore immunophenotyping at both the population and single-cell level, with more granularity than was previously available, providing greater opportunities for discovery than ever before.”

Expert tips to optimize immunophenotyping

The following list of tips is by no means exhaustive, but summarizes the points made here and includes further helpful advice from all five contributors to this editorial. Additional tips can be found in an earlier article.

• Understand the biology of your system; thoroughly review the available literature and never overlook the value of antibody vendors’ datasheets
• Identify appropriate markers before selecting suitable fluorophores
• Begin with a panel that includes basic markers for multiple immune cell types, then refine it to home in on the cells of interest
• Simplify immune cell analysis by depleting red blood cells from whole blood or tissue isolated cell suspensions
• Consider including CD45 in staining panels as a pan-leukocyte marker to exclude non-immune cells during the gating process
• Pair weakly expressed targets or very rare cell types with bright fluorophores, and vice versa
• Distribute dyes with overlapping spectra on different cells
• Fix cells before intracellular staining to ensure the target protein is retained in the original cellular location
• Some epitopes may no longer be accessible after fixation; test for compatibility if unsure and add antibodies prior to fixation if necessary
• Detecting intracellular antigens involves permeabilizing cells before staining; if using a reversible permeabilizing agent (e.g. saponin), be sure to include it in every wash step and antibody staining mix
• When using multiple intracellular markers, identify fixation/permeabilization conditions that give the best results for your markers of interest
• Test different antibody concentrations (and incubation times/temperatures) to find conditions that provide the best signal-to-noise ratio
• Always include controls and treat these identically to the test samples; unstained controls, isotype controls, single-stained compensation controls, FMO controls, and a viability stain are essential
• Use label-specific compensation controls for tandem dyes to yield more accurate results
• Know your flow cytometer; every instrument is unique