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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.

Figure 1. Initial gating of 22–color T cell panel focused on recognizing gamma delta T, conventional CD4 and CD8 T cells and T reg populations. Gating starts with singlets (top left), then live cells (top middle left), then lymphocytes gated by scatter populations (top center) to identify gamma delta T cells (top middle right) and their memory subpopulations by CD45RA and CCR7 (top right). Conventional CD4 and CD8 T cells are gated (bottom right) as well as CD25+ CD127– T regs (bottom middle) and CD45RA vs CD39 expression on these cells (bottom left). (Click image to enlarge)

Figure 2. Gating on CD4 T cell populations using 22–color T cell panel showing CD4 and CD8 expression (top left), CD4 naïve, central and effector memory populations as defined by CCR7 and CD45RA populations (top middle), follicular helper cells as defined by CXCR5 expression, (bottom left), effector memory and naïve subpopulations as defined by CD27 and CD28 expression (bottom middle), gating of T stem cell memory using CD28 and CD95 on CCR7 expressing CD4 T cells (bottom right), and PD–1 and CD38 expression on CD4 T cell subpopulations (top right) (Click image to enlarge).

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.

Figure 3. Gating on NK and NK T cell populations using 22–color T cell panel shows identification of NK and T cells (top left), CD56 expression on T cells (top middle) and CD4 and CD8 distribution of CD56+ T cells (top right). CD56 and CD16 expression is shown for CD3– cells (bottom left) as well as NKp46 on NK cell subpopulations (bottom right) (Click image to enlarge).

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. 

Cell type Grouping Product
T cell Primary CD3 Monoclonal Antibody (UCHT1), NovaFluor Blue 660–120S, eBioscience
CD4 Monoclonal Antibody (SK3), NovaFluor Blue 555, eBioscience
CD8a Monoclonal Antibody (OKT8), NovaFluor Blue 585, eBioscience
BD OptiBuild™ BUV615 Mouse Anti–Human γδ TCR Brilliant™ Ultra Violet 615 (BD Cat. No. 751308)
Secondary CD25 Monoclonal Antibody (CD25–4E3), PE–Cyanine7, eBioscience
CD27 Monoclonal Antibody (O323), NovaFluor Yellow 590, eBioscience
CD28 Monoclonal Antibody (CD28.2), NovaFluor Yellow 660, eBioscience
CD45RA Monoclonal Antibody (MEM–56), PerCP
CD127 Monoclonal Antibody (eBioRDR5), Super Bright 702, eBioscience
Brilliant Violet 785™ Anti–human CD197 (CCR7) (BL Cat. No. 353230)
Tertiary CD38 Monoclonal Antibody (HIT2), eFluor 450, eBioscience
BD OptiBuild™ BUV563 Mouse Anti–Human CD39, Brillant™ Ultra Violet 563 (BD Cat. No. 748473)
BD OptiBuild™ BUV395 Rat Anti–Human (CD185) CXCR5, Brillant™ Ultra Violet 395 (BD Cat. No. 740266)
CD194 (CCR4) Monoclonal Antibody (D8SEE), PE, eBioscience
CD196 (CCR6) Monoclonal Antibody (R6H1), Super Bright 436, eBioscience
CD279 (PD–1) Monoclonal Antibody (EH12.2H7), APC
NK cell Primary CD16 Monoclonal Antibody (3G8), NovaFluor Yellow 730, eBioscience
CD56 (NCAM) Monoclonal Antibody (TULY56), Brilliant Ultra Violet 737, eBioscience
Secondary CD335 (NKp46) Monoclonal Antibody (9E2), PerCP–eFluor 710, eBioscience
NK & T cell Tertiary CD95 (APO–1/Fas) Monoclonal Antibody (DX2), Super Bright 600, eBioscience
CD183 (CXCR3) Monoclonal Antibody (CEW33D), PE–eFluor 610, eBioscience
Live Primary LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation
All Blocking buffer Brilliant Stain Buffer
Blocking buffer CellBlox Plus Blocking Buffer
Beads UltraComp eBeads Plus Compensation Beads
Staining Buffer eBioscience Flow Cytometry Staining Buffer

Order human natural killer and T cell immunophenotyping panel 22–color products

Cy™ is a trademark or registered trademark of GE Healthcare.

Not for resale. Super Bright Polymer Dyes are sold under license from Becton, Dickinson and Company.

Brilliant Ultra Violet™ and Brilliant Violet™ are trademarks or registered trademarks of Becton, Dickinson and Company or its affiliates, and is used under license.

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