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Updated: Aug 22, 2025
High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues
Published on: September 15, 2021
Flow Cytometry Analysis of Immune Cell Responses
Kavita R Manhas1, Joseph N Blattman2
1Center for Immunotherapy, Vaccines, and Virotherapy, Biodesign Institute, Arizona State University, Tempe, AZ, USA.
Insights
This study details methods for isolating murine immune cells and analyzing T cell populations using flow cytometry. Researchers can characterize CD4+ and CD8+ T cells and their expression of chemokine receptor 7 (CCR7).
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Flow cytometry is a key technique for immune cell analysis.
- Characterizing immune cell subsets requires precise isolation and staining methods.
- Understanding T cell populations and their homing receptors is crucial in immunology.
Purpose of the Study:
- To provide a detailed protocol for isolating murine immune cells from lymphoid organs and blood.
- To describe the flow cytometric analysis of CD4+ and CD8+ T cell populations.
- To explain the measurement of chemokine receptor 7 (CCR7) expression on T cells.
Main Methods:
- Isolation of immune cells from murine inguinal lymph nodes, spleen, thymus, and peripheral blood.
- Staining of isolated cells with fluorescently tagged primary antibodies against specific immune cell markers.
- Flow cytometric analysis to quantify T cell populations (CD4+, CD8+) and CCR7 expression.
Main Results:
- Successful isolation and preparation of murine immune cells for flow cytometry.
- Quantification of CD4+ and CD8+ T cell subsets from various tissues.
- Assessment of CCR7 expression levels on T lymphocytes, indicating their lymph node homing potential.
Conclusions:
- The described methods enable robust characterization of murine T cell populations via flow cytometry.
- This protocol facilitates the study of immune cell trafficking and function by measuring CCR7 expression.
- The methodology is adaptable for analyzing other immune cell subsets and proteins of interest.
Abstract:
Flow cytometry is a fluorescence-based technology that allows for the identification and characterization of immune cell subsets within a heterogenous population. Briefly, isolated immune cells are stained in suspension with fluorescently tagged antibodies to identify cells of interest prior to being run through a flow cytometer. Here we describe how to isolate murine immune cells from various body regions, including the inguinal lymph nodes (ILNs), spleen, thymus, and peripheral blood, and tag them with primary fluorescent antibodies for flow cytometric analysis of CD4+ and CD8+ T cell populations. This chapter also details how to use flow cytometry to measure T cell expression of chemokine receptor 7 (CCR7), the major chemokine receptor lymphocytes use to enter lymph nodes. The methods described in this chapter can be used for characterizing other proteins of interest, as well as other immune cell subsets.
