Castillo-Hair, Sebastian M.Sexton, John T.Landry, Brian P.Olson, Evan J.Igoshin, Oleg A.Tabor, Jeffrey J.2016-07-152016-07-152016Castillo-Hair, Sebastian M., Sexton, John T., Landry, Brian P., et al.. "FlowCal: A User-Friendly, Open Source Software Tool for Automatically Converting Flow Cytometry Data from Arbitrary to Calibrated Units." <i>ACS Synthetic Biology,</i> 5, no. 7 (2016) American Chemical Society: 774-780. http://dx.doi.org/10.1021/acssynbio.5b00284.https://hdl.handle.net/1911/90926Flow cytometry is widely used to measure gene expression and other molecular biological processes with single cell resolution via fluorescent probes. Flow cytometers output data in arbitrary units (a.u.) that vary with the probe, instrument, and settings. Arbitrary units can be converted to the calibrated unit molecules of equivalent fluorophore (MEF) using commercially available calibration particles. However, there is no convenient, nonproprietary tool available to perform this calibration. Consequently, most researchers report data in a.u., limiting interpretation. Here, we report a software tool named FlowCal to overcome current limitations. FlowCal can be run using an intuitive Microsoft Excel interface, or customizable Python scripts. The software accepts Flow Cytometry Standard (FCS) files as inputs and is compatible with different calibration particles, fluorescent probes, and cell types. Additionally, FlowCal automatically gates data, calculates common statistics, and produces publication quality plots. We validate FlowCal by calibrating a.u. measurements of E. coli expressing superfolder GFP (sfGFP) collected at 10 different detector sensitivity (gain) settings to a single MEF value. Additionally, we reduce day-to-day variability in replicate E. coli sfGFP expression measurements due to instrument drift by 33%, and calibrate S. cerevisiae Venus expression data to MEF units. Finally, we demonstrate a simple method for using FlowCal to calibrate fluorescence units across different cytometers. FlowCal should ease the quantitative analysis of flow cytometry data within and across laboratories and facilitate the adoption of standard fluorescence units in synthetic biology and beyond.engThis is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by the American Chemical Society.Journal articlecalibrated gene expression unitsflow cytometryfluorescent proteinmolecules of equivalent fluorophorehttp://dx.doi.org/10.1021/acssynbio.5b00284