Background Spread Due to Spillover Not Corrected

Symptom

Even after compensation, background spread from spillover effects makes it difficult to determine appropriate gate boundaries. Positive and negative populations are not clearly separated in the detector of interest.

1. 1 Compensation removes spillover signals but does not correct background spread caused by spillover
2. 2 FMO (fluorescence minus one) controls not used to determine background from other fluorochromes
3. 3 Gate boundaries set without understanding cumulative background from all other channels
4. 4 High background from multiple fluorochromes accumulating in the target detector

1. 1 Use FMO controls with all antibodies in the panel minus the one being measured to determine background spread
2. 2 Prepare FMO control for each detector where gate boundary is difficult to establish
3. 3 Analyze FMO data to understand background contribution from all other fluorochromes in the target detector
4. 4 Set appropriate gates based on FMO control data showing cumulative background effects

Related Video (3)

BD Biosciences ★ 92

Flow Cytometry Compensation Tips and Tricks

"Directly addresses flow cytometry compensation strategies and practical tips, which is essential for understanding why compensation alone cannot correct background spread from spillover effects"

Bilibili (China-Accessible Mirrors) ★ 78

FlowJo Data Analysis Software Operation

"Covers FlowJo data analysis including gating strategies post-acquisition, relevant for understanding how to work with and address spillover-related background issues in detector gates"

Bilibili (China-Accessible Mirrors) ★ 72

Flow Cytometry Complete Workflow: Sample to Analysis

"Complete workflow including troubleshooting component provides context for understanding where spillover compensation failures occur and how they manifest in analysis phase"