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Defining iNKT Cell Subsets and Their Function by Flow Cytometry.

Marianthi Gioulbasani1, Ageliki Tsagaratou1,2,3

  • 1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

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Summary

This study details methods for analyzing invariant natural killer T (iNKT) cell subsets. It covers characterization by transcription factors, surface markers, and cytokine production, both in vitro and in vivo, for immune response regulation.

Keywords:
cytokinesflow cytometryiNKT cellssubsetstranscription factors

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Area of Science:

  • Immunology
  • Cell Biology
  • Flow Cytometry

Background:

  • Invariant natural killer T (iNKT) cells are crucial immune regulators.
  • iNKT cells can be functionally subdivided based on transcription factors and cytokine production.
  • Assessing these subsets is key to understanding immune responses.

Purpose of the Study:

  • To describe robust methods for characterizing iNKT cell subsets.
  • To enable detailed analysis of iNKT cell function and subsets.
  • To provide protocols for both ex vivo and in vivo assessments.

Main Methods:

  • Characterization of murine iNKT subsets using flow cytometry based on transcription factor (e.g., PLZF, RORγt) expression.
  • Identification of iNKT subsets via surface marker expression for downstream molecular assays (RNA-seq, ATAC-seq, WGBS).
  • Functional assessment through in vitro activation (PMA/ionomycin) and in vivo activation (α-galactosyl-ceramide) followed by cytokine analysis (IFNγ, IL-4).

Main Results:

  • Established protocols for distinguishing iNKT cell subsets based on transcription factors and surface markers.
  • Demonstrated methods for assessing iNKT cell cytokine production following both in vitro and in vivo activation.
  • Provided a comprehensive approach for analyzing iNKT cell subsets across various tissues.

Conclusions:

  • The described methods allow for detailed characterization of iNKT cell subsets.
  • These protocols facilitate the study of iNKT cell function in immune regulation.
  • The approaches support downstream molecular analyses for deeper insights into iNKT cell biology.