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T Lymphocyte Immunophenotyping Using Multicolor Flow Cytometry


Cluster of differentiation (CD) antigens are a complex and highly organized collection of surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication, sensing the microenvironment and in adaptive immunity. During lymphocyte maturation, immune cells will express complex variations of CD antigens on their cell surface, some of which are lost, while others are acquired at various stages either by interactions with antigen presenting cells (APC) or with other cells of the immune system. In flow cytometric immunophenotyping, monoclonal antibodies targeting CD antigens facilitate the identification and characterization of leukocytes and lymphocyte sub-populations. By using different combinations of CD antibodies, it is possible to characterize the cell surface immunophenotypes of different leukocyte sub-populations, including the functionally distinct mature cell subpopulations of B cells, helper T cells (TH), cytotoxic T cells (TC), and natural killer (NK) cells, as well as to facilitate the identification of new biomarkers and therapeutic targets of immunological and hematological diseases.



Lymphocytes express on their surface a distinct constellation of molecules, many of which are used to characterize the several stages of their lineage-specific differentiation or their different states of activation or inactivation. As surface antigens, CD molecules function in a number of different ways, often acting as receptors or ligands important to cell-cell communication. These receptors process and convey external signals into the cell by activating complex signaling cascades which ultimately alter the behavior of the cell (e.g. promote growth, differentiation, and isotype switching or cytokine production). Some CD antigens do not play a role in cell signaling, but have other functions, including adaptive immunity, cell adhesion, and platelet adhesion and platelet aggregation.

Over the past decades, CD antigens have become attractive and reliable markers for the identification and characterization of lymphocyte populations and sub-populations, a technique referred to as immunophenotyping. This method utilizes fluorochrome-labeled monoclonal antibodies (mAbs) reactive against CD markers, and together with the advances in multicolor flow cytometry, have been paramount in determining their expression and function. However, because the type of CD antigen and the degree to which it is expressed varies drastically amongst lymphocyte populations, using a combination of CD antibodies can maximize the resolution of a particular maker lineage or sub-lineage population (See table 1). In biomedical research, characterizing the dynamic expression of CD antigens in correlation to different pathophysiological conditions has become instrumental in the diagnosis and treatment of leukemia's, lymphomas and immune system disorders such as autoimmune disease. For example, the therapeutic mAb orthoclone OKT3 (also known as Muromonab-CD3) is an immunosuppressive agent used to reduce acute rejection in patients receiving organ transplants such as liver or kidney.

Table 1. Common CD markers used for the differentiation of leukocytes by flow cytometry.

Cell Type
Common Human CD Markers
Common Mouse CD Markers
T CellCD3, CD4, CD8CD3, CD4, CD8
B CellCD19, CD20CD45R/B220, CD19, CD 22 (B cell activation marker)
Dendritic CellCD11c, CD123CD11c, CD123
Natural Killer (NK) CellCD56CD335 (NKp46)
Stem Cell/PrecursorCD34 (hematopoietic stem cell only)CD34 (hematopoietic stem cell only)
Monocyte/MacrophageCD14, CD33CD11b/Mac-1, Ly-71 (F4/80)
GranulocyteCD66bCD66b, Gr-1/Ly6G, Ly6C
PlateletCD41, CD61, CD62CD41, CD61 (Integrin Β3), CD9, CD62P (activated platelets)
ErythrocyteCD235aCD235a, Ter-119
Endothelial CellCD146CD146 MECA-32, CD106, CD31, CD62E (activated endothelial cells)


T Lymphocytes and Their Subsets

T lymphocytes, which originate in the bone marrow and then migrate to the thymus for maturation, are key players of the adaptive immune system. Together with B lymphocytes, these cells orchestrate a variety of immune-related functions (e.g. eliminating infected host cells, activating additional immune cells, producing cytokines, secreting antibodies, etc.) that ultimately shape the landscape of the immune response. Differentiating T lymphocyte populations and subsets is rather straightforward since many key surface markers have already been conveniently identified and a broad selection of CD antibodies validated for their detection are commercially available.

Identifying Cytotoxic and Helper T Cell Populations

CD3, which is T cell co-receptor involved in the activation of two major T cell populations, cytotoxic T cells and helper T cells, is universally expressed on all mature T lymphocytes (CD3+). The specificity of CD3 for T lymphocytes and its appearance throughout T cell maturation makes it a defining feature and identifying marker of the T cell lineage. In tandem with the T cell receptor (TCR), CD3 will recognize and bind to either a peptide-MHC class I or class II complex on antigen presenting cells (APCs). Along with the TCR/CD3 complex, each of the two major T cell populations expresses co-receptors which bind to the peptide-MHC complex and enhance T-cell responses. CD4 co-receptors, which are expressed on the surface of helper T cells and facilitates their activation, binds to peptide-MHC class II complexes, whereas CD8 co-receptors, which are expressed on the surface of cytotoxic T cells, binds to peptide-MHC class I complexes. Through immunophenotyping, fluorescently-labeled CD antibody conjugates can be used to identify the expression of these co-receptors and distinguish between these two major T cell populations. CD3+CD4+CD8- counts are used to characterize helper T cells, and CD3+CD4-CD8+ counts are used to characterize cytotoxic T cells.

Illustration of CD4 and CD8 T cell activation (figure created in BioRender).


Table 2. Overview of Monoclonal Anti-Human CD3, CD4, and CD8 antibodies

Species Reactivity
Host, Isotype
Anti-human CD3 Recognizes CD3?-chainMonoclonalHumanMouse, IgG1HIT3b
Anti-human CD3 Recognizes CD3?-chainMonoclonalHumanMouse, IgG1, kappaSK7
Anti-human CD3 Recognizes CD3?-chainMonoclonalHumanMouse, IgG1, kappaUCHT1
Anti-human CD3 Recognizes CD3?-chainMonoclonalHumanMouse, IgG2a, kappaHIT3a
Anti-human CD3 Recognizes CD3?-chainMonoclonalHumanMouse, IgG2a, kappaOKT-3
Anti-human CD4 Recognizes CD4MonoclonalHumanMouse, IgG1, kappaRPA-T4
Anti-human CD4 Recognizes CD4MonoclonalHumanMouse, IgG1, kappaSK3
Anti-human CD4 Recognizes CD4MonoclonalHumanMouse, IgG2b, kappaOKT-4
Anti-human CD4 Recognizes CD4MonoclonalHumanMouse, IgG2b, kappaHIT4a
Anti-human CD8Recognizes CD8?MonoclonalHumanMouse, IgG1, kappa HIT8a


Designing a Multicolor Flow Cytometry Panel for T Lymphocyte Immunophenotyping

In the following section, we describe how to design a multicolor flow cytometry panel to differentiate CD4+ T cells and CD8+ T cells from peripheral blood mononuclear cell populations (PBMCs) using the following fluorescently-labeled CD antibodies: a PerCP anti-human CD3 antibody (Cat No. 100331T1), an iFluor® 488 anti-human CD4 antibody (Cat No. 10042050) and a PE anti-human CD8 antibody (Cat No. 100811L1). Factors that were instrumental in designing this 3 color panel include instrument configuration, fluorophore properties and the degree of CD marker expression.

Flow Cytometer Configuration and Fluorophore Selection

When designing a multicolor panel for flow cytometry it is important to check your instrument configuration as it can affect what fluorophores are available to you and how much compensation may be required before analyzing data. In multicolor flow cytometry, compensation is often necessary to account for the spectral overlap between fluorophores and correct for any spillover (i.e. when a signal from one fluorophore spills into the detectors of other fluorophores). While modern technology has made the compensation process much easier, choosing the right fluorophore can also minimize the need to perform compensation to begin with. To help determine fluorophore compatibility use a fluorescence spectra viewer to compare your instrument's configuration against the excitation and emission parameters of possible fluorophores.

Representation of excitation and the overlapping emission spectra of iFluor® 488 (Green, Cat No. 1023) PE (Yellow, Cat No. 2556) and PerCP (Cat No. 2559). Multicolor analysis may require compensation prior to data analysis. Compensation accounts for this overlap by removing signals that "spillover" into the main signal of interest.

The aforementioned 3-color panel was designed for an ACEA NovoCyte flow cytometer (ACEA Biosciences) equipped with a 488 nm laser and the appropriate filter sets (e.g. 530/30 nm, 585/40 nm and 670/40 nm filter sets). Using AAT Bioquest's interactive spectrum viewer, fluorophores iFluor® 488, PE, and PerCP were analyzed for spectral compatibility (Figure 1). Based on this configuration, all three fluorophores can be well-excited by the 488 nm laser, however, compensation will be required to account for iFluor® 488 spillover into the PE channel and PE spillover into the APC channel (Table 3).

Table 3. 3-Color multicolor flow cytomtery panel.
Fluorophore Ex (nm) Em (nm) Laser Peak Intensity Filters Spillover
(530/30 filter)
(585/40 filter)
(670/40 filter)
iFluor® 488 491 nm 516 nm Blue laser 96% 530/30 63% 16% 0%
PE 566 nm 574 nm Blue laser 65% 585/40 0% 79% 5%
PerCP 477 nm 678 nm Blue laser 87% 670/40 0% 0% 80%

Balance CD Marker Expression & Fluorophore Brightness

Another important parameter to consider when performing multicolor flow cytometry is to match the expression levels of the CD markers you wish to detect with an appropriately bright fluorophore. Primary CD markers are well-characterized and often densely expressed. These types of markers pair well with dim to moderately bright fluorophores. Tertiary markers, however, are poorly characterized and expressed at low levels, and pair well with moderate to bright fluorophores. In the case of CD3, CD4 and CD8 markers, which are all well-characterized, T lymphocytes have highly heterogenous CD3 expression compared to CD4 and CD8. Thus antibodies conjugated to PerCP, a relatively dim fluorophore, can be used to detect CD3. For CD4 and CD8 detection, iFluor® 488 and PE conjugated CD antibodies can be used, respectively. iFluor® 488 is a moderately bright fluorophore and PE is a bright fluorophore.

For more information regarding fluorophore brightness, refer to our application note: Relative Brightness of Fluorescent Dyes


Materials and Methods

PBMC Cell Preparation

Cryopreserved human PBMCs (10 million cells/vial, iXCells Biotechnologies Cat No. 10HU-003) were thawed and cultured immediately in order to retain the highest cell viability. To thaw cells, place vial in a 37 °C water bath with gentle agitation for ?1 minute. Pipette cells into a 15 mL conical tube with 10 mL fresh Blood Cell Culture Medium containing 10% Fetal Bovine Serum (i.e. Total Volume = 10.1 mL (100 µL PBMCs + 9 mL fresh Blood Cell Culture Medium + 1 mL Fetal Bovine Serum)). Then centrifuge at 1,000 rpm (?220g) for 5 minutes at room temperature and remove supernatant.

PBMC Cell Staining

Prior to immunolabeling, determine PBMC health using a viability assay such as the Trypsan Blue Dye Exclustion Test (AAT Bioquest, Cat No. 2452). For each staining condition we recommend using 0.5-1x106 cells/sample. Wash PBMCs with HHBS buffer (AAT Bioquest, Cat No. 20011) 2 times by centrifuge at 1000 RPM for 5 minutes each wash. Block non-specific Fc-mediated interactions by re-suspending PBMCs in Assay Buffer (HHBS buffer containing 1% BSA solution) with Human TruStain FcX™ (BioLegend, Cat No. 422302) 5 µL/100 µL of Assay Buffer and incubate at room temperature for 10 minutes. Incubate PBMCs with CD3/CD4/CD8 conjugate for 20 minutes in the dark, on ice. Wash PBMCs with Assay Buffer twice and then resuspend PBMCs in Assay buffer. Sample is now ready to be analyzed on a flow cytometer equipped with a 488 nm laser.


Product Ordering Information


Table 3. Product Ordering Information For Immunophenotyping

Ex/Em (nm)
Unit Size
Cat No.
PerCP Anti-human CD3 AntibodyHIT3a477/678 nm25 Tests100301T0
PerCP Anti-human CD3 AntibodyHIT3a477/678 nm100 Tests100301T1
PerCP Anti-human CD3 AntibodyHIT3a477/678 nm500 Tests100301T2
PerCP Anti-human CD3 AntibodyHIT3b477/678 nm25 Tests100311T0
PerCP Anti-human CD3 AntibodyHIT3b477/678 nm100 Tests100311T1
PerCP Anti-human CD3 AntibodyHIT3b477/678 nm500 Tests100311T2
PerCP Anti-human CD3 AntibodyUCHT1477/678 nm25 Tests100321T0
PerCP Anti-human CD3 AntibodyUCHT1477/678 nm100 Tests100321T1
PerCP Anti-human CD3 AntibodyUCHT1477/678 nm500 Tests100321T2
PerCP Anti-human CD3 AntibodySK7477/678 nm25 Tests100331T0