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Published on: March 5, 2019
Multiparametric Flow Cytometry Panel for Characterization of Mouse T Cell Differentiation and NK Cell Maturation
Tim Bozic1, Bostjan Markelc1,2, Simona Kranjc Brezar1,3
1Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska cesta 2, SI-1000 Ljubljana, Slovenia.
Methods and Protocols
|June 25, 2026
Summary
This study presents an optimized 14-color flow cytometry protocol for characterizing mouse lymph node immune cells. The method enables detailed analysis of T and NK cell subsets, crucial for understanding immune responses.
Area of Science:
- Immunology
- Cell Biology
Background:
- Lymph nodes are critical for immune regulation, coordinating adaptive immune responses.
- Understanding the composition and activation of lymph node immune cells is vital for assessing immune status.
- Comprehensive immunophenotyping is essential for dissecting immune organization and cellular heterogeneity.
Purpose of the Study:
- To develop and optimize a multiparametric flow cytometry protocol for characterizing mouse lymph node immune cell populations.
- To enable reliable identification and quantification of major lymphoid subsets, including T and NK cells.
- To provide a reproducible platform for immunophenotyping under various experimental conditions, including post-lipopolysaccharide challenge.
Main Methods:
- Utilized a 14-color multiparametric flow cytometry approach.
- Developed an optimized protocol involving lymph node isolation via anatomical landmarks, mechanical dissociation, and enzymatic digestion for single-cell suspension.
- Established a specific flow cytometry panel and gating strategy for T cell (CD4+, CD8+) and NK cell subset analysis.
Main Results:
- Successfully generated high-quality single-cell suspensions from mouse lymph nodes.
- Enabled reliable identification and quantification of CD4+ T helper cells, CD8+ cytotoxic T cells (including differentiation states), and NK cells (including maturation stages).
- Demonstrated the protocol's utility in assessing lymphocyte maturation following lipopolysaccharide (LPS) challenge.
Conclusions:
- The presented protocol offers a robust and reproducible method for the comprehensive immunophenotyping of T and NK cell subsets in mouse lymphoid tissues.
- This optimized flow cytometry approach is valuable for studying immune cell dynamics and heterogeneity in various experimental settings.
- The protocol facilitates a deeper understanding of immune regulation and adaptive immune responses orchestrated within lymph nodes.
