Background Spread Due to Spillover Not Corrected (Flow Cytometry (Controls))

Question

Accepted Answer

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. Common causes: 1) Compensation removes spillover signals but does not correct background spread caused by spillover 2) FMO (fluorescence minus one) controls not used to determine background from other fluorochromes 3) Gate boundaries set without understanding cumulative background from all other channels 4) High background from multiple fluorochromes accumulating in the target detector Solutions: 1) Use FMO controls with all antibodies in the panel minus the one being measured to determine background spread 2) Prepare FMO control for each detector where gate boundary is difficult to establish 3) Analyze FMO data to understand background contribution from all other fluorochromes in the target detector 4) Set appropriate gates based on FMO control data showing cumulative background effects