Flow Cytometry Immunophenotyping: Markers and Panel Design Guide

Author: Mike Blundell | Reviewer: Chloe Fenton

Immunophenotyping is the identification and classification of cells based on the expression of specific cell surface or intracellular markers using techniques such as flow cytometry.

This page focuses on how immunophenotyping is applied in experiments, including marker selection and multicolor panel design.

For an overview of principles, applications, and instrumentation, see: Immunophenotyping overview and applications .

How Immunophenotyping Is Applied in Flow Cytometry

The most common use of flow cytometry is in the identification of markers on cells, particularly those of the immune system, which is often referred to as immunophenotyping. It is the desire to detect an increased number of cell subsets of the immune system that has driven the increase in multicolor flow cytometry.

Immunophenotyping (Figure 32) can be simply identifying a cell by a single marker or more complex identification of cells, using homing profiles, activation states, and cytokine release all in one panel. As a consequence, experimental protocols are often a combination of surface and intracellular staining. In addition to basic research, immunophenotyping is now routinely used in clinical applications to diagnose disease or to monitor and evaluate residual disease.

Table 1. An example of common markers used to identify immune cells. 

A simple example of immunophenotyping using surface staining is shown below where a simple four-color staining panel can help identify four major cell subsets in human peripheral blood using markers that require no compensation. Additional information in a more complex panel may include the subsets of T cells, e.g., T helper, T cytotoxic, and T regulatory cells, using markers such as CD4, CD8, CD25, CD127, and FOXP3, whether they are naïve, memory, or activated cells, using markers such as CD45RA, CD45RO, CD62L, CD69, and HLA-DR, and determining their cytokine profile, by measuring factors such as IFN-g, IL-2, IL-17, and IL-9. It is easy to see how an 18-color panel can be quickly built.

Refer to Chapter 6 for more information on building multicolor panels. 

Fig. 32. Immunophenotyping of whole blood. Simple 4-color immunophenotyping panel using CD3, CD19, CD66b, and CD14 to identify T cells, B cells, granulocytes, and monocytes, respectively.

Multicolor Panel Design Considerations

When designing immunophenotyping panels, consider the following factors:

• Fluorophore compatibility and spectral overlap
• Marker expression levels (bright versus dim antigens)
• Instrument configuration and detector availability
• Appropriate controls for accurate gating and analysis

Table 2. The typical uses of panels of different sizes. 

Once an immunophenotyping panel has been designed and optimized, the next step is to implement the experiment using a structured workflow that ensures consistent and reliable data generation. The following workflow outlines the key stages involved in preparing samples, staining cells, and analyzing results.

Immunophenotyping Workflow

1. Prepare cell samples (e.g., blood or cell suspensions from tissues)
2. Stain with fluorophore-conjugated antibodies
3. Acquire data using a flow cytometer
4. Apply compensation and gating strategies
5. Analyze populations based on marker expression

Frequently Asked Questions