Autofluorescence Complicating Cell Population Gating Strategy (Flow Cytometry (Autofluorescence))

Question

Accepted Answer

Difficult to establish clear gates between positive and negative populations. Autofluorescent cells appear in unexpected regions of scatter plots, creating ambiguous boundaries and potential misidentification of cell populations. Common causes: 1) Heterogeneous autofluorescence levels across different cell types within sample 2) Multiple autofluorescent populations (monocytes, activated cells) overlapping with marker-positive cells 3) Lack of proper controls (unstained, FMO) to define autofluorescence baseline 4) Complex samples with mixed cell types showing variable autofluorescence intensity Solutions: 1) Use additional markers to confirm population identity when autofluorescence complicates gating 2) Include unstained controls essential for identifying autofluorescence baseline 3) Implement FMO controls to distinguish true marker signal from background in each channel 4) Apply multiparameter gating strategies using markers in less-affected channels (far-red) 5) Consider spectral cytometry with autofluorescence reference for complex gating scenarios