title: Guide to red fluorescent proteins and biosensors for flow cytometry.
abstract: Since the discovery of the first red fluorescent protein (RFP), named DsRed, 12 years ago, a wide pallet of red-shifted fluorescent proteins has been cloned and biotechnologically developed into monomeric fluorescent probes for optical microscopy. Several new types of monomeric RFPs that change the emission wavelength either with time, called fluorescent timers, or after a brief irradiation with violet light, known as photoactivatable proteins, have been also engineered. Moreover, RFPs with a large Stokes shift of fluorescence emission have been recently designed. Because of their distinctive excitation and fluorescence detection conditions developed specifically for microscopy, these fluorescent probes can be suboptimal for flow cytometry. Here, we have selected and summarized the advanced orange, red, and far-red fluorescent proteins with the properties specifically required for the flow cytometry applications. Their effective brightness was calculated for the laser sources available for the commercial flow cytometers and sorters. Compatibility of the fluorescent proteins of different colors in a multiparameter flow cytometry was determined. Novel FRET pairs, utilizing RFPs, RFP-based intracellular biosensors, and their application to a high-throughput screening, are also discussed.
authors.affiliation: Department of Anatomy and Structural Biology, and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: Kiryl D
authors.initials: KD
authors.lastname: Piatkevich
authors.forename: Vladislav V
authors.initials: VV
authors.lastname: Verkhusha
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry*; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein