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. Author manuscript; available in PMC: 2021 Mar 19.
Published in final edited form as: Curr Protoc Cytom. 2017 Apr 3;80:9.12.1–9.12.20. doi: 10.1002/cpcy.17

Fluorescent Proteins for Flow Cytometry

Teresa S Hawley 1, Robert G Hawley 2, William G Telford 3
PMCID: PMC7974382  NIHMSID: NIHMS1670341  PMID: 28369764

Abstract

Fluorescent proteins have become standard tools for cell and molecular biologists. The color palette of fluorescent proteins spans the ultraviolet, visible, and near-infrared spectrum. Utility of fluorescent proteins has been greatly facilitated by the availability of compact and affordable solid state lasers capable of providing various excitation wavelengths. In theory, the plethora of fluorescent proteins and lasers make it easy to detect multiple fluorescent proteins simultaneously. However, in practice, heavy spectral overlap due to broad excitation and emission spectra presents a challenge. In conventional flow cytometry, careful selection of excitation wavelengths and detection filters is necessary. Spectral flow cytometry, an emerging methodology that is not confined by the “one color, one detector” paradigm, shows promise in the facile detection of multiple fluorescent proteins. This chapter provides a synopsis of fluorescent protein development, a list of commonly used fluorescent proteins, some practical considerations and strategies for detection, and examples of applications.

Keywords: fluorescent proteins, conventional flow cytometry, spectral flow cytometry, lasers

INTRODUCTION

The 2008 Nobel Prize in Chemistry was awarded to Osamu Shimomura, Martin Chalfie, and Roger Tsien for the discovery and development of the green fluorescent protein, (GFP; Chalfie, 2009; Shimomura, 2009; Tsien, 2009). That recognition underscores the usefulness of fluorescent proteins (FPs) in life sciences and biomedical research (Chudakov, Matz, Lukyanov, & Lukyanov, 2010; Lippincott-Schwartz & Patterson, 2003; Tsien, 1998; Verma, 1996). The first FP, GFP, was discovered in a jellyfish belonging to class Hydrozoa in the phylum Cnidaria (Shimomura, Johnson, & Saiga, 1962). Subsequently, other FPs have been identified in coral belonging to class Anthozoa in the phylum Cnidaria (Matz et al., 1999), copepod belonging to the phylum Arthropoda (Shagin et al., 2004), lancelet, a predecessor to vertebrates belonging to the phylum Chordata (Deheyn et al., 2007), eel, a vertebrate belonging to the phylum Chordata (Kumagai et al., 2013), and bacteria (Shu et al., 2009). Class Anthozoa in the phylum Cnidaria contains the greatest diversity of FP colors. All FPs are ~25 kD in size, compared to <1 kD for organic fluorophores such as fluorescein or Texas Red. Interestingly, even though they are from diverse organisms, FPs share the unique feature of not requiring any additional enzymes or cofactors for chromophore formation; only molecular oxygen is required. There are two exceptions: The first is the recently discovered UnaG, a green FP from eel muscle fibers which requires the membrane-permeable heme metabolite, bilirubin (Kumagai et al., 2013); the second is the near-infrared (NIR) FPs derived from bacterial phytochromes, which require another heme metabolite, biliverdin (Shu et al., 2009). In both exceptions, molecular oxygen is not required.

The discovery and purification of GFP by Osamu Shimomura from the bioluminescent jellyfish Aequorea victoria in 1962 launched the FP field (Shimomura et al., 1962). It began to garner much attention after its molecular cloning by Douglas Prasher in 1992 (Prasher, Eckenrode, Ward, Prendergast, & Cormier, 1992), and the first demonstration of its utility as a fluorescent tag for in vivo labeling in the nematode Caenorhabditis elegans and bacteria by Martin Chalfi and colleagues in 1994 (Chalfie, Tu, Euskirchen, Ward, & Prasher, 1994). In 1996, two groups demonstrated the utility of GFP in monitoring gene transfer and expression in mammalian cells (Cheng, Fu, Tsukamoto, & Hawley, 1996; Levy, Muldoon, Zolotukhin, & Link, 1996). They used GFP variants with excitation maxima at 490 nm, introduced them into human fibroblasts using retroviral vectors, and detected fluorescence by microscopy as well as flow cytometry.

Current available mutagenesis has not succeeded in shifting the emission peak of GFP beyond 540 nm. A breakthrough in the red fluorescent protein field occurred in 1999 when Mikhail Matz and coworkers discovered DsRed from the non-fluorescent coral Discosoma striata, and other red fluorescent and chromoproteins from Anthozoa species found in the Indo-Pacific region (Matz et al., 1999). These discoveries paved the way for the development of orange, red, and far-red FPs with emission peaks extending as far as 655 nm (Matz, Lukyanov, & Lukyanov, 2002).

However, the vast majority of natural FPs and chromoproteins cloned from various species during the past 15 years are tetramers (such as FPs from Anthozoa and Copepoda) or dimers (such as FPs from Hydrozoa). Generation of a monomeric red FP was accomplished by Roger Tsien’s group in 2002 (Campbell et al., 2002). Subsequently, they derived a series of monomeric “Fruit” FPs, including mHoneydew, mBanana, mOrange, tdTomato, mTangerine, mStrawberry, and mCherry using site-directed mutagenesis (Shaner et al., 2004). They also derived a far-red variant named mPlum using iterative somatic hypermutation (Wang, Jackson, Steinbach, & Tsien, 2004). Intense molecular evolution of the FP chromophores continued and yielded other desirable changes, such as reduced cytotoxicity. As high-order aggregation is the most likely cause of cytotoxicity, Benjamin Glick’s group generated highly soluble tetrameric variants from DsRed in 2008 (Strack et al., 2008). DsRed-Express2 and its derivatives, E2-Orange, E2-Red/Green, and E2-Crimson, have similarly low aggregation values as enhanced GFP (EGFP). They are non-cytotoxic in bacteria and mammalian cells even when expressed at high levels.

Until recently, the conventional red laser (630 to 640 nm) that is ubiquitous on commercial flow cytometers has not been useful for FPs because it cannot efficiently excite the majority of red and far-red FPs. Only two far-red FPs, E2-Crimson and TagRFP657, can be adequately excited by conventional red lasers. The scenario changed with the development of the NIR FPs, IFP1.4 and IFP2.0 (Shu et al., 2009; Yu et al., 2014) as well as iRFP670, iRFP682, iRFP702, iRFP713, and iRFP720 (Filonov et al., 2011; Shcherbakova and Verkhusha, 2013). BphP1-FP (Shcherbakova et al., 2015) and iRFP713/V256C (Stepanenko et al., 2016), the newest members in this category, exhibit the highest fluorescence quantum yield and the highest effective brightness in mammalian cells, respectively, among available NIR FPs. These are products of extensive mutagenesis of the bacterial phytochrome photosensory receptors. In vivo imaging that benefits from the use of FPs with excitation and emission spectra falling within the so-called “optical window” (~650 to 900 nm) prompted their development (Marx, 2014). Within this window, mammalian tissues show low autofluorescence as well as low absorption by melanin, hemoglobin, and water. Some of the NIR FPs are optimally excited by conventional red lasers and have become useful in flow cytometry. As mentioned earlier, they use biliverdin as the chromophore. Indeed, sustained expression of IFP1.4 requires frequent administration of exogenous biliverdin; on the other hand, the five iRFPs efficiently incorporate endogenous biliverdin present in mammalian tissues, making them as easy to use as other FPs.

Currently, the color palette of useful FPs spans the ultraviolet, visible, and near-infrared spectra (Table 9.12.1). They include Long Stokes Shift (LSS) FPs with >100 nm separation between their excitation and emission maxima. With violet excitation, LSS FPs emit in the green, yellow, orange, or red regions of the visible spectrum. Due to the short duration of illumination, optical highlighters such as photoactivatable, photoconvertible, and photoswitchable FPs can be detected but not activated using lasers on a flow cytometer. They are used more frequently in microscopy and imaging than flow cytometry (Shcherbakova, Subach, & Verkhusha, 2012). Many FPs and FP expression vectors are available from non-profit and commercial sources (Table 9.12.2). Numerous websites provide useful information pertaining to FPs (see examples listed in Table 9.12.3).

Table 9.12.1.

Commonly Used Fluorescent Proteins

Fluorescent protein Excitation maximum (nm) Emission maximum (nm) Feasible lasers (commonly available; nm) Feasible filtersa (commonly available; nm)
UV color
 Sirius 355 424 355, 375 424/44
Blue color
 EBFP2 383 448 375, 405 450/50
 Azurite 383 450 375, 405 450/50
 mTagBFP2 399 454 375, 405 450/50
 mKalama1 385 456 375, 405 450/50
 TagBFP 402 457 375, 405 450/50
Cyan color
 Aquamarine 430 474 405, 458 450/50, 485/22
 SCFP3A 433 474 405, 458 450/50, 485/22
 mTurquoise 434 474 405, 458 450/50, 485/22
 mTurquoise2 434 474 405, 458 450/50, 485/22
 ECFP 439 475 405, 458 450/50, 485/22
 Cerulean 433 475 405, 458 450/50, 485/22
 mCerulean3 433 475 405, 458 450/50, 485/22
 CyPet 435 477 405, 458 450/50, 485/22
 TagCFP 458 480 405, 458 450/50, 485/22
 AmCyan 458 489 405, 458 450/50, 485/22
 mTFP1 462 492 405, 458 450/50, 485/22
 mMidorishi-Cyan 470 496 405, 458 450/50, 485/22
Green color
 mUKG 483 499 488 510/20, 530/30
 TurboGFP 482 502 488 510/20, 530/30
 Verdi GFP 491 503 488 510/20, 530/30
 Azami Green 492 505 488 510/20, 530/30
 ZsGreen 493 505 488 510/20, 530/30
 ZsGreen1 493 505 488 510/20, 530/30
 TagGFP2 483 506 488 510/20, 530/30
 hrGFP 500 506 488 510/20, 530/30
 hrGFP2 500 506 488 510/20, 530/30
 EGFP 488 507 488 510/20, 530/30
 Emerald 487 509 488 510/20, 530/30
 mWasabi 493 509 488 510/20, 530/30
 Superfolder GFP 485 510 488 510/20, 530/30
 Clover 505 515 488 510/20, 530/30
 mNeonGreen 506 517 488 510/20, 530/30
 Monster Green 505 518 488 510/20, 530/30
 Renilla GFP 472 540 488 510/20, 530/30
Yellow color
 TurboYFP 508 524 488, 514, 532 530/30, 550/30
 TagYFP 508 524 488, 514, 532 530/30, 550/30
 EYFP 513 527 488, 514, 532 530/30, 550/30
 Topaz 514 527 488, 514, 532 530/30, 550/30
 SYFP2 5l5 527 488, 514, 532 530/30, 550/30
 Venus 515 528 488, 514, 532 530/30, 550/30
 Citrine 516 529 488, 514, 532 530/30, 550/30
 YPet 517 530 488, 514, 532 530/30, 550/30
Yellow-Orange color
 mPapaya1 530 541 514, 532 550/30, 576/26, 585/42
Orange color
 mKO 548 559 532, 561 576/26, 585/42
 E2-Orange 540 561 532, 561 576/26, 585/42
 mOrange 548 562 532, 561 576/26, 585/42
 mOrange2 549 565 532, 561 576/26, 585/42
 mKOk 551 563 532, 561 576/26, 585/42
 mKO2 551 565 532, 561 576/26, 585/42
Red color
 mNectarine 558 578 561 585/42, 610/20
 dTomato 554 581 561 585/42, 610/20
 tdTomato 554 581 561 585/42, 610/20
 DsRed, DsRed2 553 583 561 585/42, 610/20
 DsRed-Express 553 584 561 585/42, 610/20
 TagRFP 555 584 561 585/42, 610/20
 TagRFP-T 555 584 561 585/42, 610/20
 mTangerine 568 585 561 585/42, 610/20
 DsRed monomer 556 586 561 585/42, 610/20
 mApple 568 592 561 585/42, 610/20
 mStrawberry 574 596 561 585/42, 610/20
 mRuby2 559 600 561 585/42, 610/20
 mRuby 558 605 561 585/42, 610/20
 Fusion Red 580 608 561 585/42, 610/20
 mCherry 587 610 561 585/42, 610/20
 mLumin 587 612 561 585/42, 610/20
Far Red color
 mRaspberry 598 625 561, 592 630/22, 660/20, 675/20
 TagFP635 (mKate) 588 633 561, 592 630/22, 660/20, 675/20
 mKate2 588 633 561, 592 630/22, 660/20, 675/20
 TurboFP635 (Katushka) 588 635 561, 592 630/22, 660/20, 675/20
 HcRed-Tandem 590 637 561, 592 630/22, 660/20, 675/20
 mPlum 590 649 561, 592 630/22, 660/20, 675/20
 eqFP650 592 650 561, 592 630/22, 660/20, 675/20
 mNeptune 600 650 561, 592 630/22, 660/20, 675/20
 mCardinal 604 659 561, 592 630/22, 660/20, 675/20
 NirFP (eqFP670) 605 670 561, 592 630/22, 660/20, 675/20
TagRFP675 598 675 561, 592 630/22, 660/20, 675/20
Far Red color adequately excited with red lasers
 E2-Crimson 611 646 561, 592, 633, 640 660/20, 675/20
 TagRFP657 611 657 561, 592, 633, 640 660/20, 675/20
Near IR color
 BphP1-FP 639 669 633, 640 660/20, 675/20
 iRFP670 643 670 633, 640 660/20, 675/20
 iRFP713/V256C 662 680 633, 640 660/20, 675/20
 iRFP682 663 682 633, 640 660/20, 675/20
 iRFP702 673 702 685, 705 720/40
 IFP1.4 684 708 685, 705 720/40
 IFP2.0 690 711 685, 705 720/40
 iRFP713 (iRFP) 690 713 685, 705 720/40
 iRFP720 702 720 685, 705 720/40
Long Stokes Shift (LSS)
 T-Sapphire 399 511 375, 405 510/20, 530/30
 mAmetrine 406 526 375, 405 510/20, 530/30
 LSS-mOrange 437 572 405, 458 576/26, 585/42
 LSS-mKate2 460 605 405, 458 585/42, 610/20
 mBeRFP 446 611 405, 458 585/42, 610/20
 mKeima Red 440 620 405, 458 610/20, 630/22
 LSS-mKate1 463 624 405, 458 610/20, 630/22
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a

Add notch filter to block off laser lines whenever necessary.

Table 9.12.2.

Non-Profit and Commercial Sources of Fluorescent Proteins and Expression Vectors

Source Description Website URL
Addgene Plasmid repository contains a variety of fluorescent protein plasmids deposited by authors of publications https://www.addgene.org/fluorescent-proteins/
Agilent Technologies hrGFP II (humanized green fluorescent protein) mammalian expression vectors http://www.genomics.agilent.com/en/Fluorescent-Protein-Expression/Vitality-hrGFP-II-Mammalian-Expression/?cid=AG-PT-129&tabId=AG-PR-1222
Allele Biotechnology mTFP1, mWasabi, mMaple, mClavGR2, pcFPs, LanYFP, LanRFP, and mNeonGreen http://www.allelebiotech.com/fluorescent-proteins
BD Pharmingen Mammalian expression vectors containing green (from Aequorea coerulescens) or red (from Discosoma sp.) FP as well as organelle-targeting sequences http://www.bdbiosciences.com/us/solrSearch?text=Aequorea+coerulescens&x=0&y=0
http://www.bdbiosciences.com/us/solrSearch?text=discosoma+sp&x=0&y=0
Clontech Laboratories AcGFP1, AmCyan1, AsRed2, mBanana, mCherry, Dendra2, DsRed2, DsRed-Express, DsRed-Monomer, E2-Crimson, GFP and GFPuv, HcRed1, mOrange, mOrange2, mPlum, mRaspberry, mStrawberry, tdTomato, ZsGreen1, ZsYellow1, PAmCherry, and Timer FP http://www.clontech.com/CN/Products/Fluorescent_Proteins_and_Reporters#
ATUM IP-free synthetic non-Aequorea fluorescent proteins https://www.atum.bio/products/protein-paintbox
Evrogen TagBFP, TagCFP, TagGFP2, TagYFP, TagRFP, FusionRed, mKate2, TurboGFP, TurboYFP, TurboRFP, TurboFP602, TurboFP635, TurboFP650, NirFP, PhiYFP; PS-CFP2, KFP-Red, PA-TagRFP, and Dendra2 http://www.evrogen.com/products/BasicFPs-app.shtml
Lonza pmaxFP-Green, pmaxFP-Yellow, and pmaxFP-Red vectors http://www.lonza.com/search-results.aspx?text=pmaxFP
MBL International Keima-Red, Kaede, Azami-Green, Kusabira-Cyan (KCy), Kusabira-Orange (KO), Midoriishi-Cyan (MiCy), Kikume, Kikume Green-Red (KikGR), Umikinoko-Green (UkG1), and Dronpa-Green (DG) http://www.mblintl.com/research/fluorescent-proteins.aspx
MP Biomedicals Super Glo GFP and BFP vectors http://www.mpbio.com/index.php?cPath=2_2000_2006_2034_2097
Nanolight Technology Ptilosarcus GFP, Renilla Reniformis GFP, and Renilla Mullerei GFP http://nanolight.com/69_NanoFuels/Green_Fluorescent_Proteins.html
Nature Technology Corporation Custom recombinant protein products http://www.natx.com/protein-products.html
Perkin Elmer The firefly luciferase transgene is fused either to the puromycin resistance gene or Green Fluorescent Protein (GFP) gene via T2A “self-cleaving” linker peptide for efficient co-expression with selection marker http://www.perkinelmer.com/product/redifect-red-fluc-gfp-cls960003?searchTerm=GFP&pushBackUrl=?searchName=GFP
Promega Monster Green and hMGFP mammalian expression vector https://www.promega.com/resources/protocols/technical-bulletins/101/monster-green-fluorescent-protein-phmgfp-vector-protocol/
Thermo Fisher Scientific EmGFP, YFP, CFP, BFP, and Cycle 3 GFP http://www.lifetechnologies.com/us/en/home/life-science/protein-biology/protein-expression/mammalian-protein-expression/fluorescent-protein-vectors.html
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Table 9.12.3.

Examples of Websites with Information on Fluorescent Proteins

Source Website URL
Fluorescent protein properties
Albert Einstein College of Medicine http://www.einstein.yu.edu/research/facilities/fluorescent/
University of California, San Francisco http://nic.ucsf.edu/FPvisualization/
Fluorescent protein literature references
Nikon http://www.microscopyu.com/references/fluorescentproteins.html
Zeiss http://zeiss-campus.magnet.fsu.edu/articles/probes/index.html
Fluorescent protein applications in imaging
Leica Microsystems http://www.leica-microsystems.com/science-lab/tag/tags/fluorescent-proteins/show/tag//pagetype/all/
Olympus http://olympus.magnet.fsu.edu/primer/techniques/confocal/confocalapplications.html
Examples of spectrum viewers
BD Biosciences http://www.bdbiosciences.com/in/research/multicolor/spectrum_viewer/index.jsp
BioLegend https://www.biolegend.com/spectraanalyzer
Chroma Technology https://www.chroma.com/spectra-viewer
Evrogen http://evrogen.com/spectra-viewer/viewer.shtml
Fluorophores.org http://www.fluorophores.tugraz.at/substance/
Leica Microsystems http://www.leica-microsystems.com/fluoscout/
Omega Optical https://www.omegafilters.com/curvomatic/
Semrock http://searchlight.semrock.com/
Thermo Fisher Scientific http://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html
Zeiss https://www.micro-shop.zeiss.com/?s=33040681187b1b&l=en&p=us&f=f&a=i
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FLOW CYTOMETRY OF FLUORESCENT PROTEINS

Derivation of FP-Expressing Cell Lines as References

Two publications in 1999 demonstrated the feasibility of simultaneous analysis of blue fluorescent protein (BFP), GFP, and yellow fluorescent protein (YFP; Zhu, Musco, & Grace, 1999) as well as cyan fluorescent protein (CFP), GFP, and YFP (Beavis & Kalejta, 1999). The discovery of DsRed and other red FPs in 1999 led to the possibility of combining more than three FPs for some applications. For example, the ability to efficiently introduce multiple genes into hematopoietic stem or progenitor cells represented an enabling technology to study the cooperation of oncogenes in leukemogenesis (Akimov, Ramezani, Hawley, & Hawley, 2005; Hawley, Telford, & Hawley, 2001a). At that time, CFP, GFP, YFP, DsRed, and HcRed constituted a feasible combination.

To facilitate the establishment of detection strategies, the authors derived FP-expressing cell lines as references. Retroviral vectors were used to introduce the fluorescent protein genes into a mouse hybridoma cell line that can be easily propagated in suspension, Sp2/0-Ag14 cells (ATCC #CRL-1581). Construction of the bicistronic retroviral vectors and generation of the producer cell lines have been previously described (Hawley et al., 2001a; Hawley, Telford, Ramezani, & Hawley, 2001b). In brief, each retroviral vector was engineered by standard recombinant DNA methodology to coexpress the fluorescent protein gene and the bacterial neomycin gene. The latter confers resistance to the neomycin analog G418. The retroviral vectors were transfected into GP+E-86 ecotropic packaging cells (ATCC #CRL-9642). Supernatant from transfected GP+E-86 cells was harvested and passed through 0.45-μm sterile filters. Filtration removed any contaminating cells from the retroviral vector particles. Sp2/0-Ag14 cells were incubated with supernatant containing enhanced CFP (ECFP), EGFP, enhanced YFP (EYFP), or DsRed retroviral vector particles for 4 hr at 37°C, in the presence of 2 μg/ml polybrene. The procedure was repeated with fresh vector supernatant. Two days later, cells expressing individual fluorescent protein genes were selected by the addition of 750 μg/ml G418. In addition, cells expressing various combinations of the four fluorescent protein genes were generated by incubation with appropriate mixtures of the retroviral vector supernatants using the same transduction protocol. A total of twelve cell lines stably expressing high levels of the four FPs, individually or in various combinations, were established.

Using 458 nm from an argon-ion laser to excite ECFP, EGFP, and EYFP, and 568 nm from a krypton-ion laser to excite DsRed as well as installing a notch filter in front of the YFP detector to block off the 568 nm laser line, the authors demonstrated proof of principle that four FPs could be analyzed simultaneously (Hawley et al., 2001b). As the argon- and krypton-ion lasers were expensive and not widely available on commercial flow cytometers, an alternative excitation scheme was devised and determined to be feasible (Hawley, Herbert, Eaker, & Hawley, 2004). In the alternative detection scheme, 407 nm was used to excite ECFP, and 488 nm was used to excite EGFP, EYFP, and DsRed (Fig. 9.12.1). High level expression enabled DsRed to be readily detected even with suboptimal excitation. Subsequently, simultaneous analysis of five FPs including HcRed was documented. As in the case of DsRed, high level expression enabled HcRed to be detected with 633 nm, a suboptimal excitation wavelength (Fig. 9.12.2).

Figure 9.12.1.

Figure 9.12.1

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Simultaneous analysis of 4 FPs using 407 nm to excite ECFP, and 488 nm to excite EGFP, EYFP, and DsRed on a FACSVantageSE (BD Biosciences). As 488 nm provides suboptimal excitation of DsRed, 561 nm should be used instead whenever possible. (A) Mixture of the single FPs: 0, C, G, Y, and R. (B) Cells co-expressing multiple FPs: CY, GY, CGY, GYR, or CGYR. (C) Mixture of 10 populations: 0, C, G, Y, R, CY, GY, CGY, GYR, and CGYR. Abbreviations: 0 = cells expressing no FP; C = cells expressing ECFP; G = cells expressing EGFP; Y = cells expressing EYFP; R = cells expressing DsRed; CY = cells co-expressing ECFP and EYFP; GY = cells co-expressing EGFP and EYFP; CGY = cells co-expressing ECFP, EGFP, and EYFP; GYR = cells co-expressing EGFP, EYFP, and DsRed; CGYR = cells co-expressing ECFP, EGFP, EYFP, and DsRed.

Figure 9.12.2.

Figure 9.12.2

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Simultaneous analysis of 5 FPs using 407 nm to excite ECFP, 488 nm to excite EGFP, EYFP, and DsRed, and 633 nm to excite HcRed on a FACSVantageSE. As 488 nm and 633 nm provide suboptimal excitation of DsRed and HcRed, respectively, 561 nm and 592 nm should be used instead whenever possible. (A) Mixture of the single FPs: 0, C, G, Y, R, and HcRed. (B) Cells co-expressing multiple FPs: RHc or CGYRHc. (C) Mixture of 12 populations: 0, C, G, Y, R, CY, GY, CGY, GYR, CGYR, RHc, and CGYRHc. Abbreviations: 0 = cells expressing no FP; C = cells expressing ECFP; G = cells expressing EGFP; Y = cells expressing EYFP; R = cells expressing DsRed; CY = cells co-expressing ECFP and EYFP; GY = cells co-expressing EGFP and EYFP; CGY = cells co-expressing ECFP, EGFP, and EYFP; GYR = cells co-expressing EGFP, EYFP, and DsRed; CGYR = cells co-expressing ECFP, EGFP, EYFP, and DsRed; RHc = cells co-expressing DsRed and HcRed; CGYRHc = cells co-expressing ECFP, EGFP, EYFP, DsRed, and HcRed.

Due to broad excitation and emission spectra, the most prominent feature of simultaneous detection of multiple FPs is the large spillover between detectors. It contributes to spillover spreading (Nguyen, Perfetto, Mahnke, Chattopadhyay, & Roederer, 2013). As some of the events may have negative computed channel values after fluorescence compensation, it is not advisable to display compensated FP data using the logarithmic transform since the log function is undefined at zero and less than zero. On a log display, all of the zero and negative values are placed into the first channel and pile up along the axis. Truncating the spread gives the erroneous impression of under-compensation. On the other hand, the biexponential (Moore and Parks, 2012; Parks, Roederer, & Moore, 2006) and HyperLog (Bagwell, 2005) transforms can display negative, zero, and positive numbers, thereby preserving the symmetry of the spread (Fig. 9.12.3).

Figure 9.12.3.

Figure 9.12.3

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Display of compensated data using different transform displays. (A) Mixture of 4 FPs analyzed with FACSDiva v6.1.3 (BD Biosciences) and displayed with the logarithmic transform. (B) Identical data analyzed with FACSDiva v6.1.3 and displayed with the biexponential transform. (C) Identical data analyzed with WinList v6 (Verity Software House) and displayed with the HyperLog transform.

Considerations for Practical Use of FPs

It has been known for twenty years that the chromophore structure of GFP is amenable to modification. Indeed, early manipulations yielded variants with improved intrinsic brightness and longer emission wavelengths (Cormack, Valdivia, & Falkow, 1996; Heim, Cubitt, & Tsien, 1995; Heim and Tsien, 1996). Soon after its discovery, DsRed was studied extensively with regard to maturation efficiency and oligomerization tendency (Baird, Zacharias, & Tsien, 2000; Gross, Baird, Hoffman, Baldridge, & Tsien, 2000). Revelation of the structural basis for red fluorescence in DsRed provided the basis for rational engineering of its chromophore (Wall, Socolich, & Ranganathan, 2000).

EGFP may still be the gold standard because of a combination of positive attributes (Yang, Cheng, & Kain, 1996). However, in each color class, there are FPs with overall desirable properties (Day and Davidson, 2009; Kremers, Gilbert, Cranfill, Davidson, & Piston, 2011; Piatkevich and Verkhusha, 2011; Shaner, Patterson, & Davidson, 2007; Shaner, Steinbach, & Tsien, 2005; Telford, Hawley, Subach, Verkhusha, & Hawley, 2012). Examples of useful FPs are: Blue FPs including EBFP2, TagBFP, and mTagBFP2; cyan FPs including mTurquoise, mTurquoise2, Cerulean, mCerulean3, and mTFP1; green FPs including EGFP, TagGFP2, Emerald, and mWasabi; yellow FPs including Venus, mVenus, Citrine, and YPet; orange FPs including mKO2, mKOk, mOrange, and E2-Orange; red FPs including mCherry, mApple, mRuby2, and TagRFP; far-red FPs including mKate2, mCardinal, TagRFP657, and E2-Crimson; LSS FPs including T-Sapphire, mAmetrine, LSS-mKate2, LSS-mOrange, and mBeRFP. Some new fluorescence resonance energy transfer (FRET) pairs, such as Clover>mRuby2 and mTurquoise>mNeonGreen, may perform better than the widely used CyPet>YPet (Nguyen and Daugherty, 2005) and SCFP3A>SYFP2 (Kremers, Goedhart, van Munster, & Gadella, 2006). As well, tdTomato (Shaner et al., 2004), Clover (Lam et al., 2012), mNeonGreen (Shaner et al., 2013) are very bright; Fusion Red is supermonomeric (Shemiakina et al., 2012); Superfolder GFP is designed to fold well even when fused to poorly folded polypeptides (Pedelacq, Cabantous, Tran, Terwilliger, & Waldo, 2006).

For practical use of FPs, it is worthwhile to consider some of their properties (Chudakov et al., 2010; Stepanenko, Verkhusha, Kuznetsova, Uversky, & Turoverov, 2008). Intrinsic brightness of an FP is defined as the product of its molar extinction coefficient and quantum yield; that is, how strongly it absorbs light at a given wavelength (per molar concentration), and how many photons it emits relative to the photons absorbed. In practice, when used on a conventional flow cytometer, the effective brightness depends on the excitation wavelength and detection filter. Therefore, it is important to optimize both. Maturation efficiency depends on several factors including temperature, oxygen concentration, expressing cell types, and fusion protein partners, but it is usually not a concern for FPs that have been codon optimized for mammalian cells because they mature rapidly at 37°C. High pH stability as indicated by low pKa is advantageous when FPs are targeted to acidic organelles; on the other hand, pH sensitivity can be exploited in some applications. Dimeric and tetrameric FPs are brighter than monomeric FPs, but are not recommended as fusion tags because they may produce artifacts such as atypical localization, disruption of normal function, or interference with signaling cascades. Even for whole-cell labeling, high level of expression of an FP that has the tendency to aggregate may lead to cytotoxicity (Strack, Keenan, & Glick, 2011).

A few suggestions can be made for the analysis of multiple FPs simultaneously: (1) compare the intrinsic brightness of each FP, (2) consider the factors affecting the effective brightness of each FP by displaying it on one of the many available spectrum viewers on the web, and trying out different combinations of lasers and filters to optimize the effective brightness of each FP while minimizing spillover between detectors, (3) ensure that none of the detection filters captures any laser light, and if necessary, use a notch filter to block off a laser line, (4) for specific applications, check FPs for maturation efficiency, pH stability, and aggregation tendency, and (5) if possible, explore emerging methodologies such as spectral flow cytometry (Nolan and Condello, 2013) and fluorescence lifetime flow cytometry (Houston, Naivar, & Freyer, 2010).

Simultaneous Detection of Multiple FPs

Due to their broad excitation and emission spectra, dealing with spectral overlap remains a challenge in the simultaneous detection of multiple FPs. In conventional flow cytometry, photons are directed to detectors according to wavelength: One color, one detector. For simultaneous detection of fluorophores with highly overlapping emission spectra, careful selection of optical filter for each fluorophore is required. Each single color control indicates the percentage of a particular fluorophore’s fluorescence signals spilling into detectors not assigned to that fluorophore. Fluorescence compensation is used to remove spillover signals in each detector and determine the fluorescence attributed to each fluorophore.

In spectral flow cytometry, photons are dispersed by prisms or gratings to an array of detectors; as a result, complete emission spectra are measured (Nolan and Condello, 2013). Obviating the need to select an optical filter for each fluorophore makes spectral flow cytometry ideal for simultaneous detection of multiple fluorophores with highly overlapping emission spectra. Each single color control produces a reference emission spectrum of each fluorophore. Autofluorescence is treated as a “color” as well. Unmixing algorithm deconvolutes the total measured signal in each sample and calculates the abundance of each fluorophore in a mixture (Novo, Gregori, & Rajwa, 2013).

It is indeed promising that two spectral flow cytometers using different platforms of detection have been used successfully to analyze multiple FPs simultaneously. The commercially available SP6800 and SA3800 spectral analyzers manufactured by Sony Biotechnology (San Jose, California) use a combination of prism array and 32-channel PMT as detection optics (Futamura et al., 2015). Figure 9.12.4 illustrates an example of simultaneous detection of EGFP, EYFP, and DsRed on the SP6800. Five NIR FPs can be similarly resolved on the SP6800 (Telford, Shcherbakova, Buschke, Hawley, & Verkhusha, 2015). A prototype spectral analyzer custom-made by John Nolan’s group at the Scintillon Institute (San Diego, California) uses a combination of holographic grating and electron multiplying charge-coupled device (EM-CCD) as detection optics (Nolan, Condello, Duggan, Naivar, & Novo, 2013). It has been used successfully for the simultaneous analysis of ECFP, EGFP, EYFP, and DsRed (J. P. Nolan, pers. comm.).

Figure 9.12.4.

Figure 9.12.4

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Simultaneous analysis of 3 FPs using 488 nm to excite EGFP, EYFP, and DsRed on a SP6800 spectral flow cytometer (Sony Biotechnology). (A) Each FP provides a reference emission spectrum. (B) Mixture of the 3 FPs before spectral unmixing with data displayed on a spectral plot. (C) Mixture of the 3 FPs after spectral unmixing with data displayed on dot plots.

LASERS FOR FLUORESCENT PROTEINS

The broad selection of fluorescent proteins now available for flow cytometry means that even simple flow cytometers can analyze at least one and maybe two or three FPs simultaneously. On the other hand, the wide variety of FPs also makes non-standard laser wavelengths useful for exciting many of the newer proteins. The lasers described below are all available on commercial instruments, although some are options only on more advanced cytometers (Telford, 2004).

Cyan 488 nm

The blue-green or cyan 488 nm wavelength remains the primary excitation source for virtually all flow cytometers. This wavelength was historically generated using argon-ion gas lasers; almost all modern flow cytometers now use solid state direct diode and diode-pumped solid state (DPSS) sources. The mutant EGFP and most other green fluorescent protein variants are excited at this wavelength, with detection in the fluorescein range (roughly 500 to 540 nm). EYFP and other yellow fluorescent protein variants, including Venus, Citrine, and YPet are also well-excited at 488 nm. As described in the Derivation of FP-Expressing Cell Lines as References section, EGFP and EYFP can be simultaneously analyzed at 488 nm; however, the spectral overlap is considerable, and optimized GFP (~510 nm) and YFP (>550 nm) filters are required to minimize compensation. Conventional fluorescein and phycoerythrin (PE) filters are usually not optimal for GFP/YFP analysis. DsRed, dTomato, and other short red FPs can also be excited at 488 nm to some degree, but only suboptimally.

Red 633 to 645 nm

Red lasers (formerly HeNe 633 nm units, more recently red laser diodes) were the first secondary lasers widely used on flow cytometers. However, until recently, they had limited utility for exciting FPs. Even relatively long red FPs like HcRed, mKate, E2 Crimson, and mNeptune are only suboptimally excited at this wavelength. Longer red FPs with excitation maxima in the 630 to 640 nm range have only recently become available, including TagRFP657 and iRFP670. These FPs readily combine with 488 nm-excited FPs for multicolor analysis.

Violet 405 nm

Violet laser diodes are now the primary source of violet laser excitation for flow cytometry, having replaced the krypton-ion laser generated 407 and 413 nm laser lines (Telford, Hawley, & Hawley, 2003; Telford et al., 2006). These lasers are small, inexpensive, and are often grouped with 488 and red laser sources on three laser cytometers. Their chief utility is exciting ECFP and other cyan fluorescent proteins, including the Cerulean series, CyPet, and TagCFP. Again, these FPs can be readily combined with 488 nm excited FPs for multicolor analysis.

Green to Yellow Laser Sources

Green to yellow laser sources range from 532 to 561 nm, and are now common fixtures on flow cytometers; they often constitute a fourth laser line along with cyan, red, and violet (Telford et al., 2005). While often installed for their superior ability to excite the phycobiliprotein PE and its tandem dyes, they also provide excellent excitation of most red FPs, ranging from shorter reds like DsRed to longer ones including HcRed and mPlum. Green to yellow excitation is far superior to cyan 488 nm excitation of DsRed and dTomato, and is absolutely essential for longer red FPs. As with red and violet lasers, spatially separated cyan and green to yellow sources allow analysis of green and red FPs with minimal spectral overlap.

Blue 440 to 450 nm

Less common on commercial instrumentation but still available as an option is the blue laser range of 440 to 450 nm. These are usually blue laser diodes, similar in semiconductor chemistry to violet. Blue laser diodes more closely match the excitation maxima of most cyan fluorescent proteins, and can be used to enhance detection sensitivity of these FPs. The blue laser range is also more optimal for excitation of longer wavelength cyan fluorescent proteins, including AmCyan, TagCFP, and mTFP1 (Telford, 2015a). While not a replacement for violet lasers, blue modules make useful additions to advanced multi-laser systems.

Blue laser diodes are also useful for simultaneous GFP/YFP excitation and detection. As mentioned in the Derivation of FP-Expressing Cell Lines as References section, EGFP and EYFP show strong spectral overlap, requiring significant compensation even with optimal filters. Blue laser diodes excite EGFP nearly as well as a cyan 488 nm module, while minimally exciting EYFP. Spatially separated blue and cyan lasers can therefore be used to excite EGFP and EYFP respectively with reduced overlap of EYFP into the EGFP range.

Orange 592 to 594 nm

Until recently, orange laser light was difficult to produce. Older large-scale cell sorters used dye head lasers to generate laser lines in the 595 to 600 nm range; orange HeNe lasers emitting at 594 nm were available but only at very low power levels. Small and powerful DPSS orange lasers in the 592 to 594 nm range are now available as options on advanced cytometers. They are useful for exciting longer red FPs including HcRed, mPlum, mKate, and mKate2. However, the sensitivity difference between this wavelength and the more common yellow 561 nm is not substantial.

Ultraviolet 355 to 375 nm

Ultraviolet (UV) lasers are necessary for a small but important group of flow cytometry applications. Older cytometers relied on water-cooled argon-ion and krypton-ion gas lasers to produce lines in the 351 to 365 nm range. Air-cooled HeCad lasers emitting at 325 nm saw some usage on smaller instruments, but had noise and lifetime issues. Smaller solid state UV lasers have now made this line available on smaller benchtop instruments. The selection of FPs excited in the UV is small, including Sirius, Azurite, and EBFP. UV-excited FPs have traditionally been very dim relative to autofluorescence background; however, recently derived UV FPs including EBFP2 and TagBFP have improved emission characteristics.

Near-infrared (NIR) Lasers

Flow cytometers can be equipped with lasers in the NIR range, including 660, 685, 705, and 730 nm (Telford, 2015b). These lasers allow analysis of the longest NIR FPs, including iRFP713 and iRFP720. Derivation of new NIR FPs is an area of intense effort, suggesting that new FPs with even longer wavelength excitation/emission characteristics will soon be available.

Laser Combinations

The cyan/red/violet “triad” is now a common laser combination even on simpler instruments; this configuration gives access to many FPs. For red FPs, the addition of a fourth green to yellow laser is essential; some smaller instrument platforms even offer a green to yellow option at the expense of red or violet wavelengths specifically for red FP analysis. These four lasers will cover most FPs and will allow simultaneous analysis of up to four or five FPs. Additional blue, UV, orange, or NIR lasers may be added for specialized FP applications.

EXAMPLES OF FLUORESCENT PROTEIN APPLICATIONS

Reporters of Gene Expression

By far, the most popular FP application in flow cytometry is using FPs as reporters of gene expression in live cells. For example, in the stem cell field, mCerulean, vexGFP, mCitrine, and mCherry have been used to monitor expression of the four reprogramming factors, OCT4, KLF4, SOX2, and c-MYC, during induction of human induced pluripotent stem cells from human fibroblasts (Papapetrou et al., 2009).

Lineage Tracing

Lineage tracing is increasingly applied to stem cell research and modeling of cellular heterogeneity in cancer. In lineage tracing, a single cell is marked in such a way that the mark is transmitted to the cell’s progeny. The “Brainbow” recombination fluorescent protein system (Livet et al., 2007) was used to generate R26R-Confetti mice (Snippert et al., 2010). The R26R-Confetti conditional allele consists of a CAG promoter, a loxP-flanked (floxed) STOP cassette serving as a transcriptional roadblock, and the “Brainbow 2.1” construct all targeted into the Gt(ROSA)26Sor locus. The “Brainbow 2.1” coding region is composed of two floxed head-to-tail tandem dimers. The first dimer contains a nuclear-localized hrGFPII and a reverse-oriented cytoplasmic monomeric YFP (mYFP). The other dimer contains a cytoplasmic tdimer2 and a reverse-oriented membrane-tethered mCerulean. When bred to mice that express Cre recombinase, Cre-mediated recombination removes the transcriptional road-block and stochastically places one of the four FPs under control of the CAG promoter, thereby indelibly marking the recombined cell and its progeny for their entire lifespan. By placing Cre under the control of a tissue-specific promoter, lineage tracing can be attained in virtually any tissue of choice. In addition, temporal control of activation can be achieved with a modified form of Cre recombinase that is non-functional until an inducing agent (such as doxycycline, tetracycline, RU486, or tamoxifen) is administered at a desired time point, for example, during embryonic development or adult life (Schepers et al., 2012).

Fluorescent Genetic Barcoding

Similar to the strategy employed in the derivation of FP-expressing cell lines described earlier, fluorescent genetic barcoding can be used to impart cell lines with inherited and distinguishable characteristics. The inherited properties of genetically fluorescent barcoded cells enable straightforward detection. Multiplexing power is increased by combining the number of distinct FPs as well as varying the fluorescence intensity of each FP in each cell. Multiplexed genetic barcoding is particularly useful for high throughput applications such as drug screening assays as it can reduce the number of screens by increasing the number of genetic barcoded populations in a mixture. Various combinations of CFP, GFP, tdTomato, E2-Crimson, and mCherry have been tested for the stability of their expression over time (Smurthwaite et al., 2014).

Visualizing Cell Cycle Progression

Dynamic color change between monomeric Azami Green (mAG) and monomeric Kusabira Orange2 (mKO) can be used to visualize cell cycle progression (Sakaue-Sawano et al., 2008). Both cdt1 and geminin are regulators of the cell cycle. During the cell cycle, these two proteins are ubiquitinated by specific ubiquitin E3 ligases which target them to the proteasome for degradation. In the G1 phase of the cell cycle, geminin is degraded, and only cdt1 tagged with the mKO is present, so nuclei are orange. In the S, G2, and M phases of the cell cycle, cdt1 is degraded, and only geminin tagged with the mAG is present, so nuclei are green. During G1/S transition, when cdt1 levels are decreasing and geminin levels are increasing, nuclei simultaneously fluoresce both colors.

Studying Mechanisms of Leukemogenesis

Although not an ideal pairing, EGFP and EYFP have been used to study the cooperation of oncogenes during leukemogenesis (Akimov et al., 2005). Two oncogenes from the human papillomavirus type 16 (HPV16) E6 and E7 genes were linked to EYFP, and the human telomerase reverse transcriptase (hTERT) was linked to EGFP. E6 and E7 genes accelerate the degradation of tumor suppressors p53 and retinoblastoma (Rb), respectively. hTERT is the catalytic subunit of telomerase, a specialized ribonucleoprotein complex that is responsible for adding telomeric DNA (repetitive TTAGGG sequences) to the ends of chromosomes to prevent shortening during replication. Cells expressing EGFP and EYFP singly and in combination were isolated by fluorescence-activated cell sorting. Whereas cells expressing hTERT alone underwent terminal differentiation, cells expressing HPV16 E6/E7 alone or in combination with hTERT were able to bypass senescence. Notably, the HPV16 E6/E7-immortalized cell lines were highly aneuploid while those concomitantly expressing hTERT exhibited near-diploid karyotypes, consistent with a role of hTERT in maintaining chromosome stability.

In Vivo Biotinylation Tagging

Flow cytometry can dovetail with downstream proteomics analysis to identify in vivo binding partners. For example, EGFP and EYFP were used to facilitate in vivo biotinylation of the homeodomain transcription factor TLX1 which was identified as an oncogene in T cell acute lymphoblastic leukemia (Riz, Hawley, & Hawley, 2011). In normal dorsal spinal cord development in the mouse, the homeobox genes Tlx1 and Tlx3 determine glutamatergic (excitatory) over GABAergic (inhibitory) cell fates. They are not normally expressed in T cells, but in T-cell acute lymphoblastic leukemia, both are frequently activated due to chromosomal translocations. In vivo biotinylation tagging has been successfully used for the isolation of protein-protein and protein-DNA complexes in mammalian cells. It is a method in which a protein of interest is tagged with a peptide that is biotinylated in vivo. To accomplish that, the Escherichia coli BirA biotin ligase is coexpressed with its target peptide in the same cell. For flow cytometric implementation, two expression vectors were generated, one encoding a fusion protein composed of BirA linked to EGFP, and the other encoding human TLX1 (the protein of interest) tagged with the BirA target peptide and linked to an IRESEYFP cassette. Cells co-expressing EGFP and EYFP were isolated by FACS and then lysed. Biotinylated TLX1, along with its in vivo binding partners, were captured on streptavidin beads and fractionated by SDS-PAGE. Gel fragments were subjected to in-gel digestion and analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDITOF) mass spectrometry. TLE1, a member of the Groucho/transducin-like Enhancer of split (Gro/TLE) family of transcriptional corepressors, was identified as one of the in vivo binding partners of TLX1.

Physiological Sensors

Some FPs are useful as physiological sensors. Sensitivity of GFP to low pH has been exploited to monitor autophagic flux in live cells (Hansen & Johansen, 2011). In mammalian cells, autophagy is regulated by nutrient availability and hormones, and has been suggested to be essential for cellular homeostasis. In addition to its homeostatic function, autophagy plays important physiological roles such as in intracellular protein quality control under normal conditions, and as a defense mechanism against bacterial pathogens or the toxic effects of aggregate-prone proteins. Even though it is a normal intracellular catabolic process, it has been implicated in human diseases. Cytoplasmic components which are destined to be recycled, such as proteins and organelles, are enclosed in a double-membrane structure called an autophagosome and delivered to the acidic lysosome for degradation. To monitor autophagic flux in live cells, a marker protein was created by fusing a tandem fusion of mCherry and GFP to LC3B, a marker of autophagy. mCherry is acid insensitive and GFP is acid sensitive. At the beginning of the autophagic process, both FPs emit fluorescence; later on, within the acidic lysosomal environment, GFP fluorescence is attenuated whereas mCherry fluorescence is not affected; at the end of the autophagic process, mCherry fluorescence is lost as well when the protein is degraded.

Some applications take advantage of protein domains that sense physiological changes. Such a domain can be grafted onto a single FP. For example, an FP linked to a protein domain that senses changes in analyte concentrations (e.g., calcium) or other parameters (e.g., pH, voltage, or hydrogen peroxide) results in altered FP fluorescence by conformational changes.

Monitoring Protein-Protein Interactions

FPs have also been used to monitor protein-protein interactions using FRET or bimolecular fluorescence complementation (BiFC). In intermolecular FRET, two proteins of interest are labeled with two spectrally distinct FPs as a donor/acceptor pair (Vereb, Nagy, & Szollosi, 2011). The donor’s emission spectrum has to overlap with the acceptor’s excitation spectrum. If the two proteins do not interact, both the donor and acceptor FPs will fluoresce independently. When the two proteins interact and physically associate, bringing the two FPs to close proximity, then excitation of the donor FP will result in fluorescence of the acceptor FP, producing a FRET signal. The donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through non-radiative dipole-dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance. Intermolecular FRET can be used to measure the intracellular dynamics of second messengers (e.g., cAMP). Intramolecular FRET utilizes an engineered linker (e.g., domain for analyte binding, protease cleavage, or phosphorylation) that undergoes a conformational change to separate or reorient the donor/acceptor FPs, resulting in abolition or alteration of FRET efficiency (Wu et al., 2006). In BiFC, two proteins of interest are fused to two non-fluorescent fragments of an FP. When the two proteins interact, the two fragments of the FP will be brought together to reconstitute the fluorescent chromophore. However, once formed, the fluorescent protein complex cannot be reversed, making it a less than ideal method for studying dynamic protein-protein interactions.

CONCLUSION

Reporters based on GFP and GFP-like proteins from other marine organisms provide valuable tools to monitor gene transfer and expression noninvasively in living cells. Unlike other bioluminescent reporters, the FP chromophore is intrinsic to the primary structure of the protein. Consequently, FP fluorescence does not require substrates or cofactors. Flow cytometry can evaluate and correlate multiple parameters simultaneously. Intense mutagenesis of the GFP and DsRed chromophores has produced a color palette of useful FPs spanning the UV, visible, and NIR spectra. Detection of various FPs has been greatly facilitated by the development of solid state lasers that are small, reliable, rugged, affordable, and easily integrated into existing instrumentation. As they are available in virtually any color, solid state lasers allow excitation of almost any fluorescent molecule. Designing detection strategies ceases to be an arduous task. However, due to the broad excitation and emission spectra of FPs, spectral overlap remains a challenge in the simultaneous detection of multiple FPs. In conventional flow cytometry, careful selection of excitation wavelengths and detection filters is necessary. Spectral flow cytometry, an emerging methodology that is not confined by the “one color, one detector” paradigm, shows much promise in the facile simultaneous detection of multiple fluorescent proteins (Futamura et al., 2015; Telford et al., 2015).

With a view toward new and evolving applications, it is becoming increasingly appreciated that extracellular vesicles play an important role in cell-cell communication. However, the underlying mechanisms by which they transmit information remain poorly understood. Tagging with multiple FPs will facilitate their purification, allowing new insights into their function in physiologic and pathologic processes (Koumangoye, Sakwe, Goodwin, Patel, & Ochieng, 2011; Mohr et al., 2015). Over the years, many clone libraries of GFP-tagged proteins in cells and organisms have been created. A recently described technique of enzyme-based GFP-tagging called APEX-GBP allows these clones to be used beyond flow cytometry and conventional microscopy for high-resolution analysis of subcellular protein distributions by electron microscopy (Ariotti et al., 2015). Finally, a recent approach to imaging RNAs in living cells, Spinach technology, uses RNA aptamers that mimic GFP to tag and monitor the RNAs of interest (Paige, Wu, & Jaffrey, 2011). The detection strategies developed to simultaneously monitor multiple FPs will no doubt prove useful in expanding the color palette of synthetic GFP chromophore analogs for live cell imaging applications (Dolgosheina et al., 2014; Filonov, Moon, Svensen, & Jaffrey, 2014).

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