Immunophenotyping in cancer research using flow cytometry

  • Unraveling complex immune responses in the cancer immunity cycle
  • Reproducible quantification and characterization of immune cell subsets by flow cytometry
  • Convenient immunophenotyping with pre-tested antibody panels

Whether immuno-oncology research, basic drug discovery research, advanced-stage clinical studies, or disease diagnostic phenotyping, immune cells are routinely analyzed. Flow cytometry has become the technology of choice for this since it provides the possibility of analyzing a large number of parameters simultaneously, quickly, and for a reasonable cost.  

Immuno-oncology research focuses heavily on immune cells implicated in the tolerance or rejection of cancer. In a cancer immunity cycle, cancer cells release inflammation markers that attract the attention of antigen-presenting cells, such as NK cells, dendritic cells, and macrophages, which in turn activate T cells. These activated T cells infiltrate the tumor, recognize tumor-specific antigens, and begin the process of killing tumor cells (Figure 1). Miltenyi Biotec offers pre-tested antibody panels for phenotyping of immune cells involved in the tumorigenesis process.

Figure 1: Cancer immunity cycle and involved immune cell subsets. Cancer cells release inflammation markers that attract the attention of antigen-presenting cells, such as NK cells, dendritic cells, and macrophages, which in turn activate T cells. These activated T cells infiltrate the tumor, recognize tumor-specific antigens, and begin the process of killing tumor cells.

Flow cytometry solutions for immunophenotyping

Flow cytometry allows identification of cellular markers through surface or intracellular staining techniques. These markers can be used to identify different cell types, which is useful in developing cell-specific therapies.

Furthermore, flow cytometry’s ability to characterize, count, and sort fluorescently labeled cells based on their phenotype affords it yet another advantage in immuno-oncological applications. Quantification and characterization of specific subsets of cell populations allows us to better ascertain the immunogenicity of a therapy, and thereby enhance our understanding of complex immune responses.

Our new and advanced flow cytometry instruments, as well as an expanded range of antibodies and fluorochromes to accompany the technique, enable more accurate phenotyping of cells, which in turn enables enhanced identification of cellular distribution in the cancer environment. 
Key features and advantages of flow cytometry include: 

  • The ability to analyze single-cell suspensions, for example immunophenotyping directly from whole-blood samples
  • Easy analysis of rare cell events with statistical significance because of the ability to acquire in excess of 4,000,000 events
  • The ability of flow cytometers to perform high-content and complex phenotyping makes this the gold-standard for immunophenotyping and immune response profiling
  • High suitability for multiplexed detection of markers of interest (intracellular, surface markers, phosphoproteins) and compatibility with high throughput
  • Appropriateness for multiplexed detection of secreted biomarker molecules, e.g., cytokines, in a multitude of biological samples, such as supernatants, blood derivatives, and tissue homogenates 

Immunophenotyping markers in immuno-oncology research

In this table you can find a common immunophenotyping markers to distinguish different cell types in immuno-oncology research. Click on the respective marker in order to find relevant antibodies for detection.

Cell typeSurface
markers
Transcription
factors
Secreted proteinsActivation markersExhaustion markersProliferation markers Apoptosis markersCell signaling markers 
T cellsCD3, CD4, CD8, CD27, CD45RA, CD62LT-bet,
GATA3
FoxP3, 
RORγt
IFNγ, IL-2, TNFα, IL-4, IL-5, IL-17, IL-10CD25, CD69, CD95, CD134, CD137, CD154PD-1, CTLA-4, LAG-3, TIM-3, TIGIT, CD244, CD272, CD278Ki-67, PCNA, Bcl-6pH2A.X, Caspase, Bcl-2p-Lck, p-ZAP70, p-PLCy2, p-ERK, p-NFκB
p-Akt, p-Syk, p-BLNK
B cellCD19, CD20, IgM, IgD, CD27, CD24Oct-2, PAX-5, Blimp-1 


 
IL-12, IFNy, IL-6, IL-10, TNFαCD69, CD25, CD80, CD86   
NK cell CD45, CD56, CD16, absence of CD3E4BP4
Eomes
T-bet
Id2
IFNy, TNFαCD161, NKp46
NKG2D
NKG2C
2B4
NKp30
NKp44
CD69 
 
PD-1, TIM-3, TIGIT  
Dendritic cells CD11c, HLA-DR, CD123, CD1c/BDCA-1BATF3, IRF4, IRF8IFNβ, IL-12, IL-6, TNFαCD80, CD86, CD40    
Macro-phagesCD16, CD64, CD68, CD71, CCR5 iNOS, IRF4 IL-18, IL-10, TGFβCD54 (ICAM1), CD69, CD206, CD163, CD80, CD86   
Tumor cells EpCAM, E-cadherin, CD133, CD44, CD200, CD90, CD144, CD34, CD31Lineage-specific cytokeratin subtypes       

Find here more in-depth information on flow cytometry analysis of cell proliferation, apoptosis, and cell signaling.

Standardizing flow cytometry assays for immunophenotyping

In a regulated environment, analysis of such complex phenotypes needs to be standardized in a reproducible fashion. You can learn more about how Miltenyi Biotec has standardized flow cytometry-based immunophenotyping using highly characterized REAfinity™ Recombinant Antibodies, immunophenotyping kits, software compatibility, and flexible flow cytometers.

MACS Handbook:

Antibodies

Multicolor flow cytometry panels for convenient immunophenotyping 

Miltenyi Biotec tested panels (MBTPs) are optimized and pre-designed panels for the immunophenotyping of different cell subsets by flow cytometry. Browse through the list below to find your panel of interest for a specific immune cell subset.

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