OMIP-070: NKp46-based 27-color phenotyping to define natural killer cells isolated from human tumor tissues

Optimised Multicolour Immunophenotyping Panel

In this OMIP (Optimised Multicolour Immunophenotyping Panel), the authors describe a 27-color phenotyping panel to study NK, or Natural Killer cells.1 The interest in NK cells began in the 1970s when it was discovered that they possess the ability to destroy tumor cells, and could play a role in controlling malignancies or tumor metastasis. Recent studies have shown that tumor progression and outcomes correlate with the presence of NK cells at the tumor site, making their study important from a therapeutic perspective.

NK cells have been difficult to study due to the limited amount of tissue that is normally available from tumor resections or biopsies. Therefore, it is very important to extract as much information as possible from a very limited sample size. This makes the single-cell approach the most suitable.

Sample preparation and flow cytometry 

The authors first prepared a high-viability single-cell suspension with minimal cell debris, which was important to be able to detect cell surface antigens. To do this, they used a tissue digestion method that combined collagenase Type II and DNase, which yielded a good population of viable mucosal mononuclear cells when used with human mucosal tissues.

For the flow cytometry experiments, the authors used a 27-color phenotypic panel to identify NK cell types. The gating strategy was designed such that it was possible to enumerate even other immune cells like B cells, T cells, and monocytes. NK cells were gated as live hematopoietic cells (Dead−,

CD45+), non B cells (CD19−), non-monocytes (CD14−), non-T cells (CD3−), non-innate lymphoid cells (ILC) (CD127−), and HLA-DR− cells.

The full list of markers in the panel based on purpose is:

  • Viability: Dead cells
  • Lymphocyte marker: CD45
  • Monocyte marker: CD14
  • B cell marker: CD19
  • T cell marker: CD3
  • Innate lymphoid cell marker: CD127
  • Exclusion/ activation marker: HLA-DR
  • NK cell marker and activating receptor: NKp46
  • NK cell marker and ADCC: CD16
  • Activating receptors: NKG2D, CD244
  • Co-simulatory receptor: CD2
  • Inhibitory receptors: NKG2A, CD161
  • Co-inhibitory receptor: TIGIT
  • Residency and activation markers: CD39, CD69
  • Residency marker: CD103
  • Migration marker: CX3CR1
  • Maturation and differentiation marker: CD57
  • Maturation marker: CD27
  • Maturation and adhesion marker: CD11b
  • Activation markers: CD38, CD25
  • Proliferation marker: Ki67
  • Cytolytic abilities marker: Granzyme B
  • Activation marker and NK cell marker for peripheral blood: CD56


The authors propose that this 27-colour marker is well optimized to identify and characterize NK cells from human tumor tissue samples treated with collagenase Type II and DNase digestion.


Learn more about the OMIP HERE


1Frutoso, M., Mair, F. and Prlic, M. (2020), OMIP‐070: NKp46‐Based 27‐Color Phenotyping to Define Natural Killer Cells Isolated From Human Tumor Tissues. Cytometry, 97: 1052-1056.
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