Deep phenotyping ILCs and T cells: A comparative analysis protocol for conventional and spectral flow cytometry

Area of Science:

• Immunology
• Cell Biology

Background:

• Innate lymphoid cells (ILCs) and T cells are crucial immune components.
• Accurate identification and characterization of these cells are vital for immunological research.
• Existing flow cytometry methods may have limitations in multiplexing and cross-species correlation.

Purpose of the Study:

• To present a detailed protocol for identifying human innate lymphoid cells (ILCs) and T cells.
• To adapt a flow cytometry panel for spectral flow cytometry analysis.
• To improve the flexibility and correlative potential of flow cytometry panels for immunological studies.

Main Methods:

• Development of a 19-plex flow cytometry panel for human peripheral blood and bone marrow.
• Optimization of antibody titration and single-stain controls.
• Adaptation of the panel for spectral flow cytometry, including detailed transfer procedures.

Main Results:

• Successful identification of human ILCs and T cells using the 19-plex panel.
• Demonstration of effective transfer from conventional to spectral flow cytometry.
• Enhanced ability to resolve complex fluorophore combinations and increase panel flexibility.

Conclusions:

• The presented protocol provides a robust method for high-parameter analysis of human ILCs and T cells.
• This approach facilitates better correlation between human studies and murine models.
• The protocol increases the utility and adaptability of flow cytometry in immunological research.