AutoSpill is a superior method for calculating spillover coefficients. The approach combines automated gating of cells, calculation of an initial spillover matrix based on robust linear regression, and iterative refinement to reduce error. Moreover, autofluorescence can be compensated out, by processing it as an endogenous dye in an unstained control. AutoSpill uses single-color controls and is compatible with common flow cytometry software. AutoSpill allows simpler and more robust workflows, while reducing the magnitude of compensation errors in high-parameter flow cytometry, by an average of 100,000-fold.
You can read the original paper in Nature Communications 2001. AutoSpill is now integrated into FlowJo v10.7 and above, and Floreada.io. To integrate AutoSpill into your R script, the original code is available on GitHub. Alternatively, try the AutoSpill web-browser.