Human Natural Killer (NK) and T Cell Panel 22–color

With our wide array of dyes, it is possible to zoom in on T cells and more deeply phenotype the population of interest. We have made use of our pre-optimized, experimentally verified 35–color immunophenotyping panel and generated an NK and T cell backbone panel. With this 22–color NK and T cell backbone panel, the most common markers are already incorporated to identify NK and T cells and then you can add the deeper markers based on the hypothesis you are testing.

Using this 22–color panel, NK and T cell populations can be subphenotyped. In Figure 1, both gamma delta T and conventional alpha beta CD4 and CD8 T cells are identified as well as naïve, central memory, effector memory and effector memory RA–re–expressing cells (EMRA) subpopulations. In addition, the CD4 regulatory T cell population is clearly gated as CD25+ CD127-. In Figure 2, CD4 conventional T cell subsets were also gated on CXCR5+ CD4 T cells to identify follicular helper T cells as well as T stem cell memory cells using CD45RA and CCR7 memory markers in conjunction with CD95 and CD28 expression. The comparative expression of PD–1 and CD38 is also shown on these CD4 T cell subsets.

Using this same panel for Figure 3, NK cell subsets were distinguished by gating on CD3– cells and using CD56 and CD16 expression along with NKp46. NKT cells were also identified as CD3+ CD56+ and are predominantly CD8 positive. This suggests that most CD56+ NKT cells identified, as predicted, were conventional CD8 T cells with NK markers, rather than CD1d–restricted NKT cells. CD1d–restricted NKT cells are typically recognized by CD1d tetramer staining and display CD4+ or CD4– CD8– double negative expression. There is room in the panel to add CD1d tetramer staining if desired to further subphenotype the cells.

Human PBMCs were stained and acquired on a 5–Laser Cytek Aurora with standard configuration, using the Cytek assay settings. CellBlox Blocking Buffer and Brilliant Stain Buffer were added to the antibody cocktail to block non–specific cell binding to NovaFluor dyes and polymer dye–dye interactions, respectively. Cells were stained with antibodies (Table 1) for at least 30 minutes at 4°C in the dark and fixed with 2% paraformaldehyde. Antibodies were titrated to determine optimal staining concentrations for cells. Single colors were generated on both cells and beads with the most optimal control (cells by default, beads if needed) used for unmixing raw data files. Spectral unmixing was performed using Cytek SpectroFlo software version 3.1.0. Analysis was performed using BD FlowJo version 10.8.1.

Table 1. Markers from 22–color 5–laser Cytek Aurora panel grouped by cell type expression for gamma delta, CD4 and CD8 conventional T cells; T regs; NK and NK T cells. Primary markers refer to lineage markers that define populations and are typically highly and clearly expressed. Secondary markers refer to higher density markers with more continuous expression that typically subphenotype populations. Tertiary markers are typically expressed at lower levels. 

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