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Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry
Published on: January 7, 2019
Investigating T cells by polychromatic flow cytometry
Enrico Lugli1, Leonarda Troiano, Andrea Cossarizza
1Department of Biomedical Sciences, Section of General Pathology, Modena, Italy.
Insights
Polychromatic flow cytometry (PFC) enables detailed analysis of immune cells by measuring up to 19 parameters. This chapter discusses PFC protocols and data interpretation for complex immunological studies.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Flow cytometry has significantly advanced immunology by enabling single-cell analysis.
- It facilitates the identification of immune cell subsets involved in immunoregulation and disease pathogenesis.
- Recent technological advancements allow for multi-parameter analysis, up to 19 at the single-cell level.
Purpose of the Study:
- To discuss protocols for performing polychromatic flow cytometry (PFC).
- To provide guidelines for the interpretation and analysis of complex PFC data.
- To highlight the utility of PFC in fine analysis of immune cell phenotypes.
Main Methods:
- Utilizing advanced flow cytometry equipment and fluorochromes.
- Implementing polychromatic flow cytometry (PFC) for simultaneous multi-parameter analysis (up to 19).
- Developing protocols for PFC execution and data handling.
Main Results:
- PFC allows for fine analysis of immune cell phenotypes, including T lymphocytes, B cell subsets, and intracellular phosphoproteome.
- The approach generates large datasets, necessitating specialized interpretation strategies.
- Established protocols and guidelines aid in managing and understanding complex PFC data.
Conclusions:
- Polychromatic flow cytometry is a powerful tool for in-depth immunological research.
- Effective data interpretation and analysis strategies are crucial for leveraging PFC's potential.
- This chapter provides essential guidance for researchers utilizing PFC in immunology.
Abstract:
Since its development, flow cytometry gave a relevant contribution to the field of Immunology. Its unique potential to analyse multiple parameters at the single cell level allowed the identification of unknown cell subsets with specific roles in immunoregulation as well as in the pathogenesis of several diseases. More recently, with the advent of new equipments and fluorochromes, the possibility exists to analyse simultaneously a large number (up to 19) of parameters in a single cell. This strategy, defined polychromatic flow cytometry (PFC), has been widely utilised in the last years for the fine analysis of immune cell phenotypes, including antigen-specific T lymphocytes, B cell subsets, and the intracellular phosphoproteome, among others. A huge amount of data can be generated by such an approach, and their interpretation could become a very complex and time-consuming task. Protocols for performing PFC will be discussed in this chapter, together with some guidelines for data interpretation and analysis.
