Innate Lymphoid Cell Gating Strategies for Flow Cytometry

Flow cytometry is essential for the study of innate lymphoid cells (ILCs). It allows multiparameter analysis of cell populations based on the expression of specific cell surface and/or intracellular molecules. Consensus gating strategies were established in collaboration with researchers in the ILCs field.^1,2 For ILC single cell suspension preparation, R&D Systems scientists used previously established protocols.^3,4

Group 1 ILCs
Natural killer cells
Mouse	CD3^- CD49b⁺ CD161⁺ NKp46⁺ CD27^high/low
Human	CD3^- CD57⁺ CD56^dim/bright CD16^dim/-

ILC1
Mouse/Human	CD34- CD127⁺ NKp44^- CD117^-

IEL
Mouse	CD45⁺ CD3 epsilon^- CD19^- CD161⁺ NKp46⁺ CD160⁺
Human	CD45⁺ CD3 epsilon^- CD19^- NKp44⁺ NKp46⁺ CD103⁺

Group 2 ILCs
ILC2
Mouse/Human	CD45⁺ CD3 epsilon^- CD4^- CD8^- CD19^- CD11b^- CD161^- CD90.2⁺ T1/ST2⁺ GATA3^high

Group 3 ILCs
LTi cells
Mouse/Human	CD4^+/- CD127⁺ Integrin alpha 4 beta 7⁺ ROR gamma t⁺ NKp46^- CCR6⁺

NCR-ILC3
Mouse	CD45⁺ CD3^- CD19^- ROR gamma t⁺ NKp46^- CD161 T-bet^+/-
Human	CD45⁺ CD3^- CD19^- CD127⁺ NKp44⁺ CCR6^- T-bet^+/-

NCR⁺ ILC3
Mouse	CD45⁺ CD3^- CD19^- ROR gamma t⁺ NKp46⁺ CD161^low/- T-bet^high
Human	CD45⁺ CD3^- CD19^- ROR gamma t⁺ CD127⁺ NKp44⁺ CCR6⁺ T-bet^high