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
1Insufficient compensation applied to correct for spectral overlap between fluorochromes
2Single-stain controls not properly measured for each fluorochrome in the panel
3Compensation calculated incorrectly or not applied during acquisition or analysis
4Multicolour panel design with fluorochromes having significant spectral overlap
Solutions
1Prepare single-stain controls using beads or cell samples for each fluorescent marker in the panel
2Use software to calculate and apply correct compensation either in real time during acquisition or afterward during analysis
3Run compensation controls before each multicolour experiment to determine degree of spillover
4Optimize panel design to minimize spectral overlap between fluorochromes when possible