Fluorescence Spillover into Secondary Detectors (Flow Cytometry (Controls))

Question

Accepted Answer

One fluorochrome's emission spectra spills over into another detector channel, creating false positive signals. Data appears contaminated with signals that do not represent true marker expression. Common causes: 1) Insufficient compensation applied to correct for spectral overlap between fluorochromes 2) Single-stain controls not properly measured for each fluorochrome in the panel 3) Compensation calculated incorrectly or not applied during acquisition or analysis 4) Multicolour panel design with fluorochromes having significant spectral overlap Solutions: 1) Prepare single-stain controls using beads or cell samples for each fluorescent marker in the panel 2) Use software to calculate and apply correct compensation either in real time during acquisition or afterward during analysis 3) Run compensation controls before each multicolour experiment to determine degree of spillover 4) Optimize panel design to minimize spectral overlap between fluorochromes when possible