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Flow Cytometry01:23

Flow Cytometry

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The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
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FlowCal: A User-Friendly, Open Source Software Tool for Automatically Converting Flow Cytometry Data from Arbitrary

Sebastian M Castillo-Hair1, John T Sexton1, Brian P Landry1

  • 1Department of Bioengineering, Rice University , 6100 Main Street, Houston, Texas 77005, United States.

ACS Synthetic Biology
|April 26, 2016
PubMed
Summary
This summary is machine-generated.

FlowCal is a new, free software tool that converts arbitrary flow cytometry units to standardized Molecules of Equivalent Fluorophore (MEF) units. This enables more accurate and reproducible gene expression analysis across different labs and instruments.

Keywords:
calibrated gene expression unitsflow cytometryfluorescent proteinmolecules of equivalent fluorophore

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Bioinformatics

Background:

  • Flow cytometry is crucial for single-cell analysis of molecular processes using fluorescent probes.
  • Data is reported in arbitrary units (a.u.) that lack standardization due to variations in probes, instruments, and settings.
  • Converting a.u. to Molecules of Equivalent Fluorophore (MEF) requires calibration particles but lacks accessible, nonproprietary tools, hindering data interpretation.

Purpose of the Study:

  • To introduce FlowCal, a novel, nonproprietary software tool for calibrating flow cytometry data.
  • To provide a user-friendly solution for converting arbitrary units (a.u.) to standardized Molecules of Equivalent Fluorophore (MEF).
  • To enhance the quantitative analysis and reproducibility of flow cytometry data in research.

Main Methods:

  • FlowCal software accepts Flow Cytometry Standard (FCS) files and is compatible with various calibration particles, probes, and cell types.
  • The tool offers both an intuitive Microsoft Excel interface and customizable Python scripts.
  • Automated data gating, statistical calculations, and publication-quality plot generation are included.

Main Results:

  • FlowCal successfully calibrated arbitrary units (a.u.) to Molecules of Equivalent Fluorophore (MEF) for E. coli superfolder GFP (sfGFP) expression across 10 gain settings.
  • Day-to-day variability in E. coli sfGFP expression measurements was reduced by 33% due to instrument drift correction.
  • S. cerevisiae Venus expression data was calibrated to MEF units, and a method for cross-cytometer calibration was demonstrated.

Conclusions:

  • FlowCal provides a convenient and accessible solution for standardizing flow cytometry data.
  • The software facilitates quantitative analysis and improves the comparability of results across different experiments and laboratories.
  • FlowCal is expected to promote the adoption of standardized fluorescence units in synthetic biology and other research fields.