Identification of Foxp3⁺ T follicular regulatory (Tfr) cells by flow cytometry
Ana Raquel Maceiras1, Luis Graca
1Instituto de Medina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Professor Egas Moniz, 1649-028, Lisbon, Portugal.
Insights
Flow cytometry enables multiparametric analysis of immune cells. This chapter details identifying Foxp3(+) follicular regulatory T (Tfr) cells, crucial for germinal center regulation, using this technology.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Flow cytometry is a key technology for multiparametric single-cell analysis, widely used for immune cell studies.
- Phenotypic analysis typically relies on fluorophore-conjugated antibodies binding to cell surface or intracellular molecules.
- Genetically modified reporter mice offer an alternative for intracellular staining, avoiding cell membrane permeabilization.
Purpose of the Study:
- To describe the identification of Foxp3(+) follicular regulatory T (Tfr) cells by flow cytometry.
- To highlight the role of Tfr cells in regulating germinal centers.
- To present flow cytometry as a method for characterizing Tfr cells in relation to T follicular helper (Tfh) cells.
Main Methods:
- Utilizing flow cytometry for multiparametric analysis of individual cells.
- Employing antibodies conjugated to fluorophores for specific molecule binding.
- Leveraging genetically modified mouse models expressing reporter genes for intracellular staining.
Main Results:
- Successful identification of Foxp3(+) Tfr cells using flow cytometry.
- Demonstration of Tfr cells' involvement in germinal center regulation.
- Characterization of Tfr cell phenotype in relation to Tfh cells.
Conclusions:
- Flow cytometry is an effective method for identifying and characterizing Tfr cells.
- Understanding Tfr cell populations is vital for studying germinal center immune responses.
- This methodology aids in the study of regulatory T cell subsets in various biological contexts.
Abstract:
Flow cytometry is a technology that allows multiparametric analysis of individual cells. As a consequence, it is among the most commonly used tools for the study of immune cells. It is useful both for the study of ex vivo cell populations isolated from experimental animals or human tissue and for characterizing the phenotype of cultured cells. The phenotypic analysis is based on antibodies associated to different fluorophores that specifically bind to key molecules. Genetically modified mouse strains that express a reporter gene under the control of a promoter of interest offer an important alternative for the staining of intracellular molecules without the need to permeabilize the cell membrane. In this chapter, we describe how Foxp3(+) follicular regulatory T (Tfr) cells, a population of regulatory T (Treg) cells related to T follicular helper (Tfh) cells and involved in the regulation of germinal centers (GC), can be identified by flow cytometry.
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