FlowCal

FlowCal converts flow cytometry arbitrary units into molecules of equivalent fluorophore (MEF) to enable quantitative, calibrated single-cell fluorescence measurements.

Key Features:

• Data calibration: Converts arbitrary units (a.u.) from flow cytometers into molecules of equivalent fluorophore (MEF).
• Calibration particles: Uses commercially available calibration particles to establish MEF scales.
• Input format: Accepts Flow Cytometry Standard (FCS) files as input.
• Automatic gating: Performs automated gating to isolate specific cell populations.
• Statistical calculations: Computes common summary statistics for flow cytometry data.
• Plotting: Generates publication-quality plots for calibrated fluorescence data.
• Compatibility: Supports various fluorescent probes and cell types.

Scientific Applications:

• Quantitative single-cell fluorescence: Enables reporting of single-cell fluorescence in MEF units for experiments using fluorescent probes.
• Cross-instrument and cross-laboratory standardization: Facilitates comparison of flow cytometry measurements across different instruments and settings.
• Synthetic biology reporter characterization: Supports characterization of genetically encoded fluorescent reporters such as superfolder GFP (sfGFP) and Venus.
• Validation studies: Has been used to calibrate E. coli expressing sfGFP and S. cerevisiae expressing Venus and to reduce day-to-day variability in replicate measurements by 33%.

Methodology:

Converts a.u. to MEF using commercially available calibration particles from FCS inputs, performs automatic gating, computes summary statistics, and generates plots.