State-of-the-art flow cytometers, with ~30 channels, make compensation increasingly difficult as the number of channels grows, due to the unavoidable overlap between emission spectra of fluorescent dyes. compensating, is a necessary preliminary step in the data analysis of multi-color flow cytometry. This results in the spillover of fluorescence to detectors different from the detector assigned to each dye (in classical flow cytometry). A key limitation with high-parameter flow cytometry, however, is the spectral overlap of fluorescent dyes 7. In our own field of immunology, high-parameter flow cytometry panels have become necessary, with multiple markers required to identify cellular lineages, major subsets, and activation markers. The development from single-color flow cytometry to ultra high-parameter flow cytometry has allowed an enormous growth in the data collected per cell. Development of novel fluorophores and advances in laser technology have provided a steady increase in the number of parameters that can be measured on state-of-the-art machines, roughly doubling each decade since the 1970s (Roederer’s Law for Flow Cytometry) 6. The diverse utility of flow cytometry has driven constant demand for an expansion in the number of parameters to be simultaneously measured. The ability to rapidly collect quantitative data from millions of single cells has driven the understanding of heterogeneity in complex cellular mixtures, and led to the development of many fluorescence-based functional assays 2, 3, 4, 5. AutoSpill allows simpler and more robust workflows, while reducing the magnitude of compensation errors in high-parameter flow cytometry.įluorescently labeled antibodies and flow cytometry have been the workhorse for single-cell data generation in many fields of biosciences since its development in the late 1960s 1. AutoSpill uses single-color controls and is compatible with common flow cytometry software. Moreover, autofluorescence can be compensated out, by processing it as an endogenous dye in an unstained control. The approach combines automated gating of cells, calculation of an initial spillover matrix based on robust linear regression, and iterative refinement to reduce error. Here, we present AutoSpill, an alternative method for calculating spillover coefficients. The calculation of spillover coefficients from single-color controls has remained essentially unchanged since its inception, and is increasingly limited in its ability to deal with high-parameter flow cytometry. Even the advent of spectral cytometry cannot circumvent the spillover problem, with spectral unmixing an intrinsic part of such systems.

Compensating in flow cytometry is an unavoidable challenge in the data analysis of fluorescence-based flow cytometry.

Flowjo 10 compensation matrix