A high-parameter spectral cytometry protocol for immune subset and functional characterization of cryopreserved human blood with 49 biomarkers.

Comprehensive immune profiling of human blood is a critical approach for evaluating immune responses following therapeutic intervention in clinical trials. Here, we present a protocol to assess leukocyte lineages, activation status, and selected immune checkpoint expression in cryopreserved buffy coat using a high-dimensional, 49-color spectral flow cytometry antibody panel. We provide instructions for thawing, washing, and staining circulating immune cells, followed by guidance on data acquisition. This protocol is suitable for studying cancer immunology and other indications requiring deep immunophenotyping.