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Updated: Aug 5, 2026
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Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications
Published on: April 14, 2015
Protocol for multiplexed quantification of fluorescent biosensor FRET efficiency using barcoded cells
Jhen-Wei Wu1, Yichu Xu1, Yun Chen2
1Department of Pathology, Johns Hopkins University, Baltimore, MD 21287, USA; Center for Cell Dynamics, Johns Hopkins University, Baltimore, MD 21218, USA.
STAR Protocols
|July 30, 2026
Summary
This study introduces a new method for simultaneously imaging multiple Förster resonance energy transfer (FRET) biosensors in live cells. The protocol enables efficient calculation of FRET efficiency for enhanced molecular activity monitoring.
Area of Science:
- Cell biology
- Biophysics
- Molecular imaging
Background:
- Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) is crucial for monitoring molecular activities in live cells.
- Existing methods often lack the capacity for simultaneous analysis of multiple FRET biosensors.
Purpose of the Study:
- To present a protocol for determining FRET efficiency using simultaneous imaging of multiple FRET biosensors.
- To enable the analysis of multiple FRET biosensors alongside donor and acceptor FPs.
Main Methods:
- Utilizing spectrally orthogonal fluorescent cell barcodes for simultaneous imaging.
- Detailed steps for cell culturing, transfection, image acquisition, and analysis.
- Calculation of FRET efficiency.
Main Results:
- Successful simultaneous imaging of multiple FRET biosensors.
- Facilitation of simultaneous analysis of multiple FRET biosensors within live cells.
- Accurate determination of FRET efficiency.
Conclusions:
- The presented protocol allows for the simultaneous analysis of multiple FRET biosensors.
- This approach enhances the monitoring of molecular activities in live cells.
- Provides a comprehensive method for FRET efficiency calculation.
