FlowCal: A User-Friendly, Open Source Software Tool for Automatically Converting Flow Cytometry Data from Arbitrary to Calibrated Units

Abstract

Flow 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, non-proprietary 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 mVenus 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.

Absolute quantification of translational regulation and burden using combined sequencing approaches

“…To date, the accuracy of models in synthetic biology has been hampered by a lack of reliable, quantitative, and high‐throughput measurements of genetic parts and devices, as well as their effects on the host cell. Attempts have been made to improve this situation by using standard calibrants to increase reproducibility across laboratories and equipment (Davidsohn et al , ; Beal et al , ; Castillo‐Hair et al , ) and by including synthetic RNA spike‐ins to enable absolute quantification of transcription (Owens et al , ). Our methodology complements these efforts by combining RNA‐seq and Ribo‐seq with RNA spike‐in standards to quantify the regulation of transcription and translation.…”

A Miniaturized Escherichia coli Green Light Sensor with High Dynamic Range

“…Next, we co‐transformed each of the three plasmids pairwise with pSR58.6, which encodes ccaR and a superfolder green fluorescent protein ( sfGFP ) reporter (Figures B and S1) . We then utilized flow cytometry to measure sfGFP expression levels under green and red light, following our previous protocols (see the Experimental Section) . We observed a CcaSR v 2.0 output of (1.06±0.12)×10 3 molecules of equivalent fluorescein (MEFL) sfGFP under red light and (1.163±0.042)×10 5 under green (Figure C).…”

“…Calibration bead samples (Spherotech RCP‐30‐5A) diluted in PBS were measured each day an experiment was conducted. After data acquisition, raw flow cytometry files were processed with FlowCal, as previously described . Specifically, cell populations were density‐gated (80 %), and the raw fluorescence values [a.u.]…”

An open-hardware platform for optogenetics and photobiology

“…Calibration beads (RCP-30-5A, Spherotech) were measured at each session. Data was processed and sfGFP and mCherry fluorescence were calibrated to Molecules of Equivalent Fluorescein (MEFL) and Molecules of Equivalent Cy5 (MECY) with FlowCal67.…”