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