Here, we present a protocol for setting three spectral flow cytometry panels for the characterization of human unconventional CD8+NKG2A/C+ T cells as well as other T and natural killer cell subsets. We describe steps for standardizing, preparing, and staining the cells, the experimental setup, and the final data analysis. This protocol should be advantageous in various settings including immunophenotyping of limited samples, immune function evaluation/monitoring, as well as research in oncology, autoimmune, and infectious diseases.
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