Deep phenotyping ILCs and T cells: A comparative analysis protocol for conventional and spectral flow cytometry
Here, we present a protocol for identifying human innate lymphoid cells (ILCs) and T cells using a 19-plex flow cytometry panel. We describe steps for titrating antibodies, preparing single-stain controls and samples, and staining cells for human peripheral blood and bone marrow samples. We then detail procedures for transferring a flow cytometry panel optimized for conventional cytometry to spectral flow cytometry analysis. This protocol facilitates correlation with murine models, maximizes measurable parameters, resolves complex fluorophore combinations, and increases panel flexibility.
Authors
- Sinisa Savic (ORCID: https://orcid.org/0000-0001-7910-0554)
- Karen Hogg (ORCID: https://orcid.org/0000-0001-7015-5102)
- Jillian L. Barlow (ORCID: https://orcid.org/0000-0002-8680-3589)
- Jadie Acklam (ORCID: https://orcid.org/0000-0003-1041-3370)
- Ian S. Hitchcock
- Catherine Cargo
- Ruth M. De Tute
- Richard Leach
- Sukhveer K. Mann
- Andy C. Rawstron
Institutions
- University of Leeds (GB)
- St James's University Hospital (GB)
- Nonwovens Innovation & Research Institute (GB)
- NIHR Leeds Musculoskeletal Biomedical Research Unit (GB)
- University of York (GB)
Publication Details
- Journal
- STAR Protocols
- Published
- 2026-07-16
- DOI
- https://doi.org/10.1016/j.xpro.2026.104669
- Primary Topic
- IL-33, ST2, and ILC Pathways
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Medical Research Council
- Leeds Biomedical Research Centre