Rare Cell Population Incorrectly Gated or Missed (Flow Cytometry (Sample Considerations))

Question

Accepted Answer

Target rare immune cell subsets (e.g., dendritic cells, innate lymphoid cells, hematopoietic progenitors) appear overestimated or masked by abundant terminally differentiated cells. Gating on single markers yields incorrect population percentages (e.g., 4.5% vs. true 1.2% DCs). Common causes: 1) Rare populations (DCs, ILCs, progenitors) are overwhelmed by abundant terminally differentiated cells in lymphoid tissues 2) Single-parameter histogram gating captures outliers (CD11c-intermediate MHCII+/-) as false positives 3) Lineage-positive cells not excluded, contaminating the rare population gate 4) Leukocytes in non-lymphoid tissues (brain, lung, liver, tumor) not distinguished from tissue-resident cells Solutions: 1) Include lineage markers (Ter119, CD3, CD19, B220, CD11b, CD11c) in a dump channel to exclude differentiated cells 2) Use bivariate dot plots or pseudocolor plots with two markers (e.g., CD11c vs. MHCII) instead of single-parameter histograms 3) Add CD45 (leukocyte common antigen) to multicolor panels when analyzing non-lymphoid tissues to identify hematopoietic cells 4) Use BioLegend mouse or human Lineage Cocktails for convenient lineage exclusion gating