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– 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. AutoSpread is available in binary form in FlowJo v.10.7 (patent pending).Ĭompensating in flow cytometry is an unavoidable challenge in the data analysis of fluorescence-based flow cytometry. To allow a large user base to take immediate advantage of the approaches reported here, an implementation of AutoSpill is included in the release of FlowJo v.10.7.
#Compensation in flowjo code
The R package also includes batch code to reproduce results as generated by the website. In addition, AutoSpill is accessible as a freely available web service at. The R package is also available in the Supplementary Information as Supplementary Data. Source code for AutoSpill is available through the R package autospill, available at the github repository 45, which includes batch code that reproduces the reported results for the datasets MM1, HS1, HS2, and Be1. Source data are provided with this paper. Note that the compensation controls for the MM2 dataset are the MM1 dataset. The raw data for the eight analyzed datasets is available at FlowRepository ( ), with IDs FR-FCM-Z2SV (Be1), FR-FCM-Z2ST (HS1 & HS2), FR-FCM-Z2SS (MM1), FR-FCM-Z2SW (MM2), FR-FCM-Z2SJ (MM3), FR-FCM-Z2SK (MM4), and FR-FCM-Z2SL (MM5).
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