Autofluorescence Complicating Cell Population Gating Strategy

Symptom

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.

1. 1 Heterogeneous autofluorescence levels across different cell types within sample
2. 2 Multiple autofluorescent populations (monocytes, activated cells) overlapping with marker-positive cells
3. 3 Lack of proper controls (unstained, FMO) to define autofluorescence baseline
4. 4 Complex samples with mixed cell types showing variable autofluorescence intensity

1. 1 Use additional markers to confirm population identity when autofluorescence complicates gating
2. 2 Include unstained controls essential for identifying autofluorescence baseline
3. 3 Implement FMO controls to distinguish true marker signal from background in each channel
4. 4 Apply multiparameter gating strategies using markers in less-affected channels (far-red)
5. 5 Consider spectral cytometry with autofluorescence reference for complex gating scenarios

Related Video (3)

BD Biosciences ★ 85

Flow Cytometry Compensation Tips and Tricks

"Directly addresses compensation and spectral overlap issues that are fundamental to managing autofluorescence artifacts in gating strategies"

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Choosing Proper Flow Cytometry Controls

"Explains proper control selection for flow cytometry experiments, essential for distinguishing autofluorescence from true positive populations"

BD Biosciences ★ 72

Cell Preparation for Flow Cytometry

"Cell preparation best practices can reduce autofluorescence variability across cell types and improve population resolution"