Effectiveness evaluation of fluorescent compensation in multicolor flow cytometry: A quantitative study

In fluorescence flow cytometry, spectral overlap among multiple fluorescent labels cannot be avoided, and thus detected fluorescent intensities need to be compensated. Although fluorescent compensation in flow cytometry has been widely used for many years, it still lacks quantitative evaluations to validate its effectiveness. Using a home-developed nine-color fluorescence flow cytometer, this study first obtains calibration curves by assaying gradient concentrations of nine different fluorochromes individually, with the fluorescent intensities of the highest concentrations of each fluorochrome being used to obtain a spillover matrix. Mixed fluorescent solutions are analyzed by flow cytometry in which the obtained fluorescent intensities are compensated by the spillover matrix, translated to specific concentrations based on calibration curve and compared with nominal values. Three mixed solutions of Brilliant Violet 650 and Brilliant Violet 711, of Alexa Fluor 488 and PE, and of Pacific Orange, Alexa Fluor 488, and PE are tested, with fluorescent compensation being observed to reduce excessive signals due to spectral overlap. Specifically, concentration deviations (before vs after compensation) in comparison with nominal values for Brilliant Violet 711 and Alexa Fluor 488 are quantified as 40.6% vs 14.9% and 6.7% vs 1.9%, respectively. The results presented here provide a quantitative reference for fluorescent compensation that can be used to effectively address the issue of spectral overlap in fluorescence flow cytometry.