期刊
ACS SYNTHETIC BIOLOGY
卷 5, 期 7, 页码 774-780出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.5b00284
关键词
flow cytometry; fluorescent protein; molecules of equivalent fluorophore; calibrated gene expression units
资金
- National Science Foundation [EFRI-1137266, MCB-1244135]
- Office of Naval Research [MURI N000141310074, YIP N000141410487]
- National Institutes of Health [1R21AI115014-01A1]
- Welch Foundation [C-1856]
- NSF Graduate Research Fellowship [DGE-0940902]
- National Defense Science and Engineering Graduate (NDSEG) Fellowship [32 CFR 168a]
- Directorate For Engineering
- Emerging Frontiers & Multidisciplinary Activities [1137266] Funding Source: National Science Foundation
- Division Of Physics
- Direct For Mathematical & Physical Scien [1427654] Funding Source: National Science Foundation
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, 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.
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