Evaluation of Methods for Identifying Human Monocytes by Flow Cytometry: TLR2 May be an Optimal Marker

Immunology. 2026 Sep 7. doi: 10.1111/imm.70195. Online ahead of print.

ABSTRACT

Monocytes play crucial roles in the pathobiology of a wide range of medical conditions, including infection, autoimmunity, cardiovascular disease, and malignancies. Studies using flow cytometry revealed the complexity of monocyte biology, including identification of three human monocyte subsets: classical (CD14^pos CD16^neg), intermediate (CD14^pos CD16^pos) and non-classical (CD14^neg/dim CD16^pos) monocytes. Still, gating strategies used for identification of monocytes by flow cytometry vary substantially between studies, which may significantly impact results and hamper reproducibility. However, to our knowledge, no previous study has evaluated several different gating strategies for identification of the total circulating monocyte population in humans. Here, healthy donor peripheral blood mononuclear cells (PBMCs, n = 10) were analysed by flow cytometry to compare seven commonly used gating strategies for defining human monocytes: (1) negative selection (CD3^neg CD19^neg CD56^neg CD66^neg), (2) FSC-SSC, (3) CD14^pos, (4) CD64^pos, (5) HLA-DR^pos, (6) CD14-HLA-DR and (7) TLR2^pos. We show that the different gating strategies significantly impacted the enumeration of non-classical monocytes, but less so for classical and intermediate monocyte populations. Compared to negative selection gating (considered the reference standard), the FSC-SSC-based gating showed the highest degree of contamination by other cell types, whereas CD14^pos, CD64^pos, CD14-HLA-DR and HLA-DR^pos gating showed highest degree of exclusion of monocytes, particularly non-classical monocytes. In comparison, TLR2^pos gating showed both the least contamination and least erroneous exclusion of monocytes when compared to negative selection gating. These results based on 'manual gating' were supported by more objective t-SNE and clustering analyses. In conclusion, TLR2^pos gating and negative selection-based gating displayed both minimal contamination and exclusion of true monocytes, with TLR2^pos gating yielding the overall purest monocyte population. Thus, adapting to more uniform gating strategies for monocyte analysis, such as a TLR2 based gating strategy, may increase data quality and reproducibility between laboratories.

PMID:42706662 | DOI:10.1111/imm.70195