A Flow Cytometry-Based Method for Profiling Immune Cells in Human Blood and Tumors

Flow cytometry enables the simultaneous characterization of multiple immune-cell populations and their phenotypic subsets in clinical specimens. However, comprehensive immunophenotyping of limited blood and tumor samples can be challenging because of limited cell availability, sample-processing requirements, and the need for standardized staining and gating procedures. Here, we describe a 12-color full-spectrum flow cytometry method for profiling major immune-cell populations in human peripheral blood and tumor tissue. The panel comprises 11 antibody markers and DAPI and is designed to identify major lymphoid and myeloid populations, including T cells, B cells, NK cells, monocytes, and granulocyte populations, with further characterization of selected cell populations based on marker expression. The workflow includes sample preparation, antibody titration, multicolor staining, viability assessment, spectral reference controls, spectral unmixing, and hierarchical gating. The protocol was optimized for relatively small clinical specimens and can be applied to both whole blood and enzymatically dissociated tumor tissue. Representative data demonstrate the ability of the panel to resolve distinct immune-cell subset distributions in individual blood and paired tumor/peri-tumor specimens. This workflow provides a practical approach for multiparameter immune profiling of limited human blood and tumor specimens and can serve as a basis for subsequent studies requiring more extensive cohort-level validation.