A standardized single-tube 17-color spectral flow cytometry workflow for integrated immunophenotyping of human PBMCs and mixed co-culture systems

We present a standardized single-tube 17-color spectral flow cytometry workflow for integrated immunophenotyping of human peripheral blood mononuclear cells (PBMCs). This Methods Article describes a quality-controlled protocol that combines lineage identification with activation, regulatory, and checkpoint-associated markers within a reproducible quality-control framework. Panel optimization incorporated spectral similarity index (SI) and spillover spreading matrix (SSM) metrics, antigen-density-guided fluorochrome assignment, systematic antibody titration, fluorescence-minus-one (FMO)-anchored gating, autofluorescence extraction, and longitudinal internal controls. The workflow supports robust immunophenotyping of PBMCs across experiments when appropriate viability gating, autofluorescence correction, and quality-control checkpoints are applied, and enables CD45-based compartment discrimination in mixed immune-tumor co-culture systems. Single-tube multiparameter staining facilitates unsupervised cluster analysis and supports identification of expected and potentially underappreciated immune subsets. The manuscript emphasizes methodological transparency, reproducibility strategies, and principles supporting panel transferability rather than hypothesis-driven biological discovery. This work provides a practical and transferable framework for high-dimensional immune monitoring in translational and preclinical settings.