FlowCal: A User-Friendly, Open Source Software Tool for Automatically Converting Flow Cytometry Data from Arbitrary to Calibrated Units.
FlowCal: A User-Friendly, Open Source Software Tool for Automatically Converting Flow Cytometry Data from Arbitrary to Calibrated Units.
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DOI：
10.1021/acssynbio.5b00284
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发表时间：
2016-07-15
影响因子：
4.7
通讯作者：
Tabor JJ
中科院分区：
文献类型：
--
作者：
Castillo-Hair SM;Sexton JT;Landry BP;Olson EJ;Igoshin OA;Tabor JJ
流式细胞术广泛用于通过荧光探针以单细胞分辨率测量基因表达和其他分子生物学过程。流式细胞仪以任意单位（a.u.）输出数据。这些随探头、仪器和设置而变化。可以使用市售的校准颗粒将任意单位转换为等效荧光团（MEF）的校准单位分子。但是，没有方便的非专有工具可用于执行此校准。因此，大多数研究人员报告的数据在a.u.，限制解释。在这里，我们报告一个名为FlowCal的软件工具，以克服目前的局限性。FlowCal可以使用直观的Microsoft Excel界面或可定制的Python脚本运行。该软件接受流式细胞术标准（FCS）文件作为输入，并与不同的校准颗粒，荧光探针和细胞类型兼容。此外，FlowCal自动门控数据，计算常见的统计数据，并产生出版质量图。我们通过校准a.u.测量E.在10个不同的检测器灵敏度（增益）设置下收集的表达超折叠GFP（sfGFP）的大肠杆菌的MEF值。此外，我们降低了重复E中的日间变异性。coli sfGFP表达测量值由于仪器漂移33%，并校准S.酿酒酵母mVenus表达数据换算成MEF单位。最后，我们展示了一个简单的方法，使用FlowCal校准不同的流式细胞仪的荧光单位。FlowCal应简化实验室内和实验室间流式细胞术数据的定量分析，并促进合成生物学及其他领域标准荧光单位的采用。
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.