Immunophenotyping of conventional and non-conventional T cell populations by surface staining of peripheral blood mononuclear cells.

Here, we present a comprehensive and reproducible protocol for the isolation, cryopreservation, and immunophenotyping of human peripheral blood mononuclear cells (PBMCs) using a 28-color flow cytometry panel. This high-dimensional panel enables the simultaneous assessment of conventional and unconventional T cells and their activation or functional states in a single sample, making it a valuable tool for translational and clinical immunology research. This protocol outlines detailed procedures for blood processing, density gradient separation, and optimized washing steps to minimise platelet contamination. Standardized cryopreservation methods are provided to facilitate longitudinal studies or multicentre trials. The staining strategy is specifically optimized for PBMCs and is compatible with both conventional and spectral flow cytometry systems, allowing robust and reproducible identification of lymphocyte populations with a focus on T lymphocytes. This method is applicable to both fresh and cryopreserved PBMCs and has been validated on material from healthy donors and patients. It provides a reliable framework for immune monitoring in contexts such as infection, cancer, or immunotherapy. Additionally, this protocol includes key troubleshooting steps, guidance on panel design, and practical advice for data acquisition and analysis. The workflow is adaptable to other staining panels and can seamlessly be integrated into standardized immune profiling pipelines.