Guide to red fluorescent proteins and biosensors for flow cytometry

Area of Science:

• Biotechnology
• Cell Biology
• Optical Microscopy

Background:

• Red fluorescent proteins (RFPs) have evolved significantly since DsRed's discovery, with engineered variants for microscopy like fluorescent timers and photoactivatable proteins.
• Recently developed RFPs with large Stokes shifts offer unique optical properties.
• Microscopy-optimized fluorescent probes are often suboptimal for flow cytometry.

Purpose of the Study:

• To select and summarize advanced orange, red, and far-red fluorescent proteins suitable for flow cytometry.
• To evaluate the effective brightness of these proteins for commercial flow cytometers and sorters.
• To determine the compatibility of different colored fluorescent proteins in multiparameter flow cytometry.

Main Methods:

• Selection and summarization of advanced orange, red, and far-red fluorescent proteins.
• Calculation of effective brightness for standard laser sources in flow cytometry.
• Assessment of fluorescent protein compatibility for multiparameter flow cytometry.

Main Results:

• Identification of optimal orange, red, and far-red fluorescent proteins for flow cytometry applications.
• Quantification of effective brightness tailored to common flow cytometry laser lines.
• Evaluation of spectral compatibility for multicolor flow cytometry panels.

Conclusions:

• Specific red-shifted fluorescent proteins are well-suited for flow cytometry, overcoming limitations of microscopy-focused probes.
• The presented data aids in selecting appropriate fluorescent proteins for enhanced multiparameter flow cytometry.
• Future applications include novel FRET pairs, biosensors, and high-throughput screening using these optimized RFPs.