37‑Color Spectral Flow Cytometry Panel for Conventional, Non‑conventional and Innate Lymphoid Cells in Human PBMC using the CytoFLEX mosaic 88 Spectral Detection Module
Executive Summary
Defining complex immune populations — particularly T cells and innate lymphoid cells (ILCs) — remains challenging due to the absence of universally accepted marker combinations. ILCs are rare in peripheral blood (0.1–1% of lymphocytes) and are primarily defined through lineage exclusion rather than unique positive markers. This application note evaluates a high‑dimensional 37‑color spectral flow cytometry panel designed to comprehensively characterize conventional T cells, unconventional T‑cell subsets (including MAIT and iNKT cells), ILCs, and natural killer (NK) cells, and further resolves B‑cell, basophil, and myeloid (monocyte, pDC) subsets in human peripheral blood mononuclear cells (PBMCs).
Samples were acquired on a CytoFLEX LX Flow Cytometer equipped with a CytoFLEX mosaic 88 Spectral Detection Module. Acquiring 5 million events per sample, combined with lineage‑exclusion gating, supported robust detection of rare populations such as ILCs and provides an empirical foundation for developing consistent gating strategies and standardized immune cell nomenclature.
Key Highlights & Findings
- Broad Phenotypic Coverage: Resolves conventional TCRαβ and TCRγδ T cells, unconventional subsets (MAIT, iNKT), ILC1/ILC2/ILC3 subsets, NK‑cell subsets (NK1–NK3), B cells, basophils, plasmacytoid dendritic cells (pDCs), and monocyte subsets.
- High‑Event Acquisition: Collecting 5 million events per sample enables robust detection of extremely rare peripheral populations such as ILCs, which represent only 0.1–1% of lymphocytes.
- Sequential Gating Logic: Employs lineage‑exclusion strategies — segregating TCRαβ from TCRγδ and excluding iNKT/MAIT prior to downstream conventional T‑cell analysis — to avoid marker interference before subtyping functional populations (e.g., Treg‑like and Th1/Th2/Th17‑like subsets).
- Experimental Impact: Overnight resting of cryopreserved PBMCs reduced pDC and basophil frequencies alongside a relative increase in intermediate monocytes, consistent with known effects of in vitro culture conditions on myeloid populations.
Note: This study also underscores the complexity of immune cell classification and the challenges of defining subsets using current phenotypic markers; high‑event acquisition is essential for reliable detection of rare populations such as ILCs.
For Research Use Only. Not for use in diagnostic procedures.
