Fluorescent Proteins for Flow Cytometry

Teresa S. Hawley1, Robert G. Hawley2, William G. Telford3

1 Flow Cytometry Core Facility, Laboratory of Genome Integrity, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, 2 Department of Anatomy and Regenerative Biology, George Washington University School of Medicine and Health Sciences, Washington, D.C., 3 Flow Cytometry Core Facility, Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.12
DOI:  10.1002/cpcy.17
Online Posting Date:  April, 2017
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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. © 2017 by John Wiley & Sons, Inc.

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

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Table of Contents

  • Introduction
  • Flow Cytometry of Fluorescent Proteins
  • Lasers for Fluorescent Proteins
  • Examples of Fluorescent Protein Applications
  • Conclusion
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

Literature Cited
  Akimov, S. S., Ramezani, A., Hawley, T. S., & Hawley, R. G. (2005). Bypass of senescence, immortalization, and transformation of human hematopoietic progenitor cells. Stem Cells, 23, 1423–1433. doi: 10.1634/stemcells.2005‐0390
  Ariotti, N., Hall, T. E., Rae, J., Ferguson, C., McMahon, K. A., Martel, N., … Parton, R. G. (2015). Modular detection of GFP‐labeled proteins for rapid screening by electron microscopy in cells and organisms. Developmental Cell, 35, 513–525. doi: 10.1016/j.devcel.2015.10.016
  Bagwell, C. B. (2005). Hyperlog‐a flexible log‐like transform for negative, zero, and positive valued data. Cytometry Part A, 64, 34–42. doi: 10.1002/cyto.a.20114
  Baird, G. S., Zacharias, D. A., & Tsien, R. Y. (2000). Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proceedings of the National Academy of Sciences U.S.A., 97, 11984–11989. doi: 10.1073/pnas.97.22.11984
  Beavis, A. J. & Kalejta, R. F. (1999). Simultaneous analysis of the cyan, yellow and green fluorescent proteins by flow cytometry using single‐laser excitation at 458 nm. Cytometry, 37, 68–73. doi: 10.1002/(SICI)1097‐0320(19990901)37:1%3c68::AID‐CYTO8%3e3.0.CO;2‐J
  Campbell, R. E., Tour, O., Palmer, A. E., Steinbach, P. A., Baird, G. S., Zacharias, D. A., & Tsien, R. Y. (2002). A monomeric red fluorescent protein. Proceedings of the National Academy of Sciences U.S.A., 99, 7877–7882. doi: 10.1073/pnas.082243699
  Chalfie, M. (2009). GFP: Lighting up life (Nobel Lecture). Angewandte Chemie (International ed. in English), 48, 5603–5611. doi: 10.1002/anie.200902040
  Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., & Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. Science, 263, 802–805. doi: 10.1126/science.8303295
  Cheng, L., Fu, J., Tsukamoto, A., & Hawley, R. G. (1996). Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells. Nature Biotechnology, 14, 606–609. doi: 10.1038/nbt0596‐606
  Chudakov, D. M., Matz, M. V., Lukyanov, S., & Lukyanov, K. A. (2010). Fluorescent proteins and their applications in imaging living cells and tissues. Physiological Reviews, 90, 1103–1163. doi: 10.1152/physrev.00038.2009
  Cormack, B. P., Valdivia, R. H., & Falkow, S. (1996). FACS‐optimized mutants of the green fluorescent protein (GFP). Gene, 173, 33–38. doi: 10.1016/0378‐1119(95)00685‐0
  Day, R. N. & Davidson, M. W. (2009). The fluorescent protein palette: Tools for cellular imaging. Chemical Society Reviews, 38, 2887–2921. doi: 10.1039/b901966a
  Deheyn, D. D., Kubokawa, K., McCarthy, J. K., Murakami, A., Porrachia, M., Rouse, G. W., & Holland, N. D. (2007). Endogenous green fluorescent protein (GFP) in amphioxus. The Biological Bulletin, 213, 95–100. doi: 10.2307/25066625
  Dolgosheina, E. V., Jeng, S. C., Panchapakesan, S. S., Cojocaru, R., Chen, P. S., Wilson, P. D., Hawkins, N., Wiggins, P. A., & Unrau, P. J. (2014). RNA mango aptamer‐fluorophore: A bright, high‐affinity complex for RNA labeling and tracking. ACS Chemical Biology, 9, 2412–2420. doi: 10.1021/cb500499x
  Filonov, G. S., Moon, J. D., Svensen, N., & Jaffrey, S. R. (2014). Broccoli: Rapid selection of an RNA mimic of green fluorescent protein by fluorescence‐based selection and directed evolution. Journal of the American Chemical Society, 136, 16299–16308. doi: 10.1021/ja508478x
  Filonov, G. S., Piatkevich, K. D., Ting, L. M., Zhang, J., Kim, K., & Verkhusha, V. V. (2011). Bright and stable near‐infrared fluorescent protein for in vivo imaging. Nature Biotechnology, 29, 757–761. doi: 10.1038/nbt.1918
  Futamura, K., Sekino, M., Hata, A., Ikebuchi, R., Nakanishi, Y., Egawa, G., … Tomura, M. (2015). Novel full‐spectral flow cytometry with multiple spectrally‐adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement. Cytometry Part A, 87, 830–842. doi: 10.1002/cyto.a.22725
  Gross, L. A., Baird, G. S., Hoffman, R. C., Baldridge, K. K., & Tsien, R. Y. (2000). The structure of the chromophore within DsRed, a red fluorescent protein from coral. Proceedings of the National Academy of Sciences U.S.A., 97, 11990–11995. doi: 10.1073/pnas.97.22.11990
  Hansen, T. E. & Johansen, T. (2011). Following autophagy step by step. BMC Biology, 9, 39. doi: 10.1186/1741‐7007‐9‐39
  Hawley, T. S., Herbert, D. J., Eaker, S. S., & Hawley, R. G. (2004). Multiparameter flow cytometry of fluorescent protein reporters. In T. S. Hawley, & R. G. Hawley (Eds.), Methods in molecular biology (Vol. 263, pp. 219–238). New York: Springer Science+Business Media. doi: 10.1385/1‐59259‐773‐4:219
  Hawley, T. S., Telford, W. G., & Hawley, R. G. (2001a). “Rainbow” reporters for multispectral marking and lineage analysis of hematopoietic stem cells. Stem Cells, 19, 118–124. doi: 10.1634/stemcells.19‐2‐118
  Hawley, T. S., Telford, W. G., Ramezani, A., & Hawley, R. G. (2001b). Four‐color flow cytometric detection of retrovirally expressed red, yellow, green, and cyan fluorescent proteins. BioTechniques, 30, 1028–1034.
  Heim, R., Cubitt, A. B., & Tsien, R. Y. (1995). Improved green fluorescence. Nature, 373, 663–664. doi: 10.1038/373663b0
  Heim, R. & Tsien, R. Y. (1996). Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Current Biology, 6, 178–182. doi: 10.1016/S0960‐9822(02)00450‐5
  Houston, J. P., Naivar, M. A., & Freyer, J. P. (2010). Digital analysis and sorting of fluorescence lifetime by flow cytometry. Cytometry Part A, 77, 861–872. doi: 10.1002/cyto.a.20930
  Koumangoye, R. B., Sakwe, A. M., Goodwin, J. S., Patel, T., & Ochieng, J. (2011). Detachment of breast tumor cells induces rapid secretion of exosomes which subsequently mediate cellular adhesion and spreading. PLoS One, 6, e24234. doi: 10.1371/journal.pone.0024234
  Kremers, G. J., Gilbert, S. G., Cranfill, P. J., Davidson, M. W., & Piston, D. W. (2011). Fluorescent proteins at a glance. Journal of Cell Science, 124, 157–160. doi: 10.1242/jcs.072744
  Kremers, G. J., Goedhart, J., van Munster, E. B., & Gadella, T. W., Jr. (2006). Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Forster radius. Biochemistry, 45, 6570–6580. doi: 10.1021/bi0516273
  Kumagai, A., Ando, R., Miyatake, H., Greimel, P., Kobayashi, T., Hirabayashi, Y., … Miyawaki, A. (2013). A bilirubin‐inducible fluorescent protein from eel muscle. Cell, 153, 1602–1611. doi: 10.1016/j.cell.2013.05.038
  Lam, A. J., St‐Pierre, F., Gong, Y., Marshall, J. D., Cranfill, P. J., Baird, M. A., … Lin, M. Z. (2012). Improving FRET dynamic range with bright green and red fluorescent proteins. Nature Methods, 9, 1005–1012. doi: 10.1038/nmeth.2171
  Levy, J. P., Muldoon, R. R., Zolotukhin, S., & Link, C. J., Jr. (1996). Retroviral transfer and expression of a humanized, red‐shifted green fluorescent protein gene into human tumor cells. Nature Biotechnology, 14, 610–614. doi: 10.1038/nbt0596‐610
  Lippincott‐Schwartz, J. & Patterson, G. H. (2003). Development and use of fluorescent protein markers in living cells. Science, 300, 87–91. doi: 10.1126/science.1082520
  Livet, J., Weissman, T. A., Kang, H., Draft, R. W., Lu, J., Bennis, R. A., … Lichtman, J. W. (2007). Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature, 450, 56–62. doi: 10.1038/nature06293
  Marx, V. (2014). Probes: Seeing in the near infrared. Nature Methods, 11, 717–720. doi: 10.1038/nmeth.3001
  Matz, M. V., Fradkov, A. F., Labas, Y. A., Savitsky, A. P., Zaraisky, A. G., Markelov, M. L., & Lukyanov, S. A. (1999). Fluorescent proteins from nonbioluminescent Anthozoa species. Nature Biotechnology, 17, 969–973. doi: 10.1038/13657
  Matz, M. V., Lukyanov, K. A., & Lukyanov, S. A. (2002). Family of the green fluorescent protein: Journey to the end of the rainbow. Bioessays, 24, 953–959. doi: 10.1002/bies.10154
  Mohr, M., Tosun, S., Arnold, W. H., Edenhofer, F., Zanker, K. S., & Dittmar, T. (2015). Quantification of cell fusion events human breast cancer cells and breast epithelial cells using a Cre‐LoxP‐based double fluorescence reporter system. Cellular and Molecular Life Sciences, 72, 3769–3782. doi: 10.1007/s00018‐015‐1910‐6
  Moore, W. A. & Parks, D. R. (2012). Update for the logicle data scale including operational code implementations. Cytometry. Part A, 81, 273–277. doi: 10.1002/cyto.a.22030
  Nguyen, A. W. & Daugherty, P. S. (2005). Evolutionary optimization of fluorescent proteins for intracellular FRET. Nature Biotechnology, 23, 355–360. doi: 10.1038/nbt1066
  Nguyen, R., Perfetto, S., Mahnke, Y. D., Chattopadhyay, P., & Roederer, M. (2013). Quantifying spillover spreading for comparing instrument performance and aiding in multicolor panel design. Cytometry. Part A, 83, 306–315. doi: 10.1002/cyto.a.22251
  Nolan, J. P. & Condello, D. (2013). Spectral flow cytometry. Current Protocols in Cytometry, 63, 1.27:1.27.1‐1.27.13. doi: 10.1002/0471142956.cy0127s63
  Nolan, J. P., Condello, D., Duggan, E., Naivar, M., & Novo, D. (2013). Visible and near infrared fluorescence spectral flow cytometry. Cytometry. Part A, 83, 253–264. doi: 10.1002/cyto.a.22241
  Novo, D., Gregori, G., & Rajwa, B. (2013). Generalized unmixing model for multispectral flow cytometry utilizing nonsquare compensation matrices. Cytometry. Part A, 83, 508–520. doi: 10.1002/cyto.a.22272
  Paige, J. S., Wu, K. Y., & Jaffrey, S. R. (2011). RNA mimics of green fluorescent protein. Science, 333, 642–646. doi: 10.1126/science.1207339
  Papapetrou, E. P., Tomishima, M. J., Chambers, S. M., Mica, Y., Reed, E., Menon, J., … Sadelain, M. (2009). Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c‐Myc expression for efficient human iPSC induction and differentiation. Proceedings of the National Academy of Sciences U.S.A., 106, 12759–12764. doi: 10.1073/pnas.0904825106
  Parks, D. R., Roederer, M., & Moore, W. A. (2006). A new “Logicle” display method avoids deceptive effects of logarithmic scaling for low signals and compensated data. Cytometry Part A, 69, 541–551. doi: 10.1002/cyto.a.20258
  Pedelacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C., & Waldo, G. S. (2006). Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology, 24, 79–88. doi: 10.1038/nbt1172
  Piatkevich, K. D., & Verkhusha, V. V. (2011). Guide to red fluorescent proteins and biosensors for flow cytometry. Methods in Cell Biology, 102, 431–461. doi: 10.1016/B978‐0‐12‐374912‐3.00017‐1
  Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., & Cormier, M. J. (1992). Primary structure of the Aequorea victoria green‐fluorescent protein. Gene, 111, 229–233. doi: 10.1016/0378‐1119(92)90691‐H
  Riz, I., Hawley, T. S., & Hawley, R. G. (2011). Lentiviral fluorescent protein expression vectors for biotinylation proteomics. In T. S. Hawley, R. G. Hawley (Eds.), Methods in molecular biology, Vol. 699 (pp. 431–447). New York: Springer Science+Business Media. doi: 10.1007/978‐1‐61737‐950‐5_21
  Sakaue‐Sawano, A., Kurokawa, H., Morimura, T., Hanyu, A., Hama, H., Osawa, H., … Miyawaki, A. (2008). Visualizing spatiotemporal dynamics of multicellular cell‐cycle progression. Cell, 132, 487–498. doi: 10.1016/j.cell.2007.12.033
  Schepers, A. G., Snippert, H. J., Stange, D. E., van den Born, M., van Es, J. H., van de Wetering, M., & Clevers, H. (2012). Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal adenomas. Science, 337, 730–735. doi: 10.1126/science.1224676
  Shagin, D. A., Barsova, E. V., Yanushevich, Y. G., Fradkov, A. F., Lukyanov, K. A., Labas, Y. A., … Matz, M. V. (2004). GFP‐like proteins as ubiquitous metazoan superfamily: Evolution of functional features and structural complexity. Molecular Biology and Evolution, 21, 841–850. doi: 10.1093/molbev/msh079
  Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N., Palmer, A. E., & Tsien, R. Y. (2004). Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnology, 22, 1567–1572. doi: 10.1038/nbt1037
  Shaner, N. C., Lambert, G. G., Chammas, A., Ni, Y., Cranfill, P. J., Baird, M. A., … Wang, J. (2013). A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nature Methods, 10, 407–409. doi: 10.1038/nmeth.2413
  Shaner, N. C., Patterson, G. H., & Davidson, M. W. (2007). Advances in fluorescent protein technology. Journal of Cell Science, 120, 4247–4260. doi: 10.1242/jcs.005801
  Shaner, N. C., Steinbach, P. A., & Tsien, R. Y. (2005). A guide to choosing fluorescent proteins. Nature Methods, 2, 905–909. doi: 10.1038/nmeth819
  Shcherbakova, D. M., Baloban, M., Pletnev, S., Malashkevich, V. N., Xiao, H., Dauter, Z., & Verkhusha, V. V. (2015). Molecular basis of spectral diversity in near‐infrared phytochrome‐based fluorescent proteins. Chemistry & Biology, 22, 1540–1551. doi: 10.1016/j.chembiol.2015.10.007
  Shcherbakova, D. M., Subach, O. M., & Verkhusha, V. V. (2012). Red fluorescent proteins: Advanced imaging applications and future design. Angewandte Chemie (International ed. in English), 51, 10724–10738. doi: 10.1002/anie.201200408
  Shcherbakova, D. M., & Verkhusha, V. V. (2013). Near‐infrared fluorescent proteins for multicolor in vivo imaging. Nature Methods, 10, 751–754. doi: 10.1038/nmeth.2521
  Shemiakina, I. I., Ermakova, G. V., Cranfill, P. J., Baird, M. A., Evans, R. A., Souslova, E. A., & Shcherbo, D. (2012). A monomeric red fluorescent protein with low cytotoxicity. Nature Communications, 3, 1204. doi: 10.1038/ncomms2208
  Shimomura, O. (2009). Discovery of green fluorescent protein (GFP) (Nobel Lecture). Angewandte Chemie (International ed. in English), 48, 5590–5602. doi: 10.1002/anie.200902240
  Shimomura, O., Johnson, F. H., & Saiga, Y. (1962). Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. Journal of Cellular and Comparative Physiology, 59, 223–239. doi: 10.1002/jcp.1030590302
  Shu, X., Royant, A., Lin, M. Z., Aguilera, T. A., Lev‐Ram, V., Steinbach, P. A., & Tsien, R. Y. (2009). Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome. Science, 324, 804–807. doi: 10.1126/science.1168683
  Smurthwaite, C. A., Hilton, B. J., O'Hanlon, R., Stolp, Z. D., Hancock, B. M., Abbadessa, D., … Wolkowicz, R. (2014). Fluorescent genetic barcoding in mammalian cells for enhanced multiplexing capabilities in flow cytometry. Cytometry Part A, 85, 105–113. doi: 10.1002/cyto.a.22406
  Snippert, H. J., van der Flier, L. G., Sato, T., van Es, J. H., van den Born, M., Kroon‐Veenboer, C., … Clevers, H. (2010). Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell, 143, 134–144. doi: 10.1016/j.cell.2010.09.016
  Stepanenko, O. V., Verkhusha, V. V., Kuznetsova, I. M., Uversky, V. N., & Turoverov, K. K. (2008). Fluorescent proteins as biomarkers and biosensors: Throwing color lights on molecular and cellular processes. Current Protein & Peptide Science, 9, 338–369. doi: 10.2174/138920308785132668
  Stepanenko, O. V., Baloban, M., Bublikov, G. S., Shcherbakova, D. M., Stepanenko, O. V., Turoverov, K. K., … Verkhusha, V. V. (2016). Allosteric effects of chromophore interaction with dimeric near‐infrared fluorescent proteins engineered from bacterial phytochromes. Scientific Reports, 6, 18750. doi: 10.1038/srep18750
  Strack, R. L., Keenan, R. J., & Glick, B. S. (2011). Noncytotoxic DsRed derivatives for whole‐cell labeling. In T. S. Hawley, R. G. Hawley (Eds.), Methods in molecular biology Vol. 699 (pp. 355–370). New York: Springer Science+Business Media. doi: 10.1007/978‐1‐61737‐950‐5_17
  Strack, R. L., Strongin, D. E., Bhattacharyya, D., Tao, W., Berman, A., Broxmeyer, H. E., & Glick, B. S. (2008). A noncytotoxic DsRed variant for whole‐cell labeling. Nature Methods, 5, 955–957. doi: 10.1038/nmeth.1264
  Telford, W. G. (2004). Small lasers in flow cytometry. In T. S. Hawley, R. G. Hawley (Eds.), Flow cytometry protocols Vol. 263 (pp. 399–418). London: Humana Press. doi: 10.1385/1‐59259‐773‐4:399
  Telford, W. G. (2015a). Near‐ultraviolet laser diodes for brilliant ultraviolet fluorophore excitation. Cytometry Part A, 87, 1127–1137. doi: 10.1002/cyto.a.22686
  Telford, W. G. (2015b). Near infrared lasers in flow cytometry. Methods, 82, 12–20. doi: 10.1016/j.ymeth.2015.03.010
  Telford, W. G., Hawley, T. S., & Hawley, R. G. (2003). Analysis of violet‐excited fluorochromes by flow cytometry using a violet laser diode. Cytometry. Part A, 54, 48–55. doi: 10.1002/cyto.a.10046
  Telford, W. G., Hawley, T., Subach, F., Verkhusha, V., & Hawley, R. G. (2012). Flow cytometry of fluorescent proteins. Methods, 57, 318–330. doi: 10.1016/j.ymeth.2012.01.003
  Telford, W., Kapoor, V., Jackson, J., Burgess, W., Buller, G., Hawley, T., & Hawley, R. (2006). Violet laser diodes in flow cytometry: An update. Cytometry Part A, 69, 1153–1160. doi: 10.1002/cyto.a.20340
  Telford, W., Murga, M., Hawley, T., Hawley, R., Packard, B., Komoriya, A., … Hubert, C. (2005). DPSS yellow‐green 561‐nm lasers for improved fluorochrome detection by flow cytometry. Cytometry Part A, 68, 36–44. doi: 10.1002/cyto.a.20182
  Telford, W. G., Shcherbakova, D. M., Buschke, D., Hawley, T. S., & Verkhusha, V. V. (2015). Multiparametric flow cytometry using near‐infrared fluorescent proteins engineered from bacterial phytochromes. PLoS One, 10, e0122342. doi: 10.1371/journal.pone.0122342
  Tsien, R. Y. (1998). The green fluorescent protein. Annual Review of Biochemistry, 67, 509–544. doi: 10.1146/annurev.biochem.67.1.509
  Tsien, R. Y. (2009). Constructing and exploiting the fluorescent protein paintbox (Nobel Lecture). Angewandte Chemie (International ed. in English), 48, 5612–5626. doi: 10.1002/anie.200901916
  Vereb, G., Nagy, P., & Szollosi, J. (2011). Flow cytometric FRET analysis of protein interaction. In T. S. Hawley, & R. G. Hawley (Eds.), Methods in molecular biology Vol. 699 (pp. 371–392). New York: Springer Science+Business Media. doi: 10.1007/978‐1‐61737‐950‐5_18
  Verma, I. M. (1996). “Green light” for gene transfer. Nature Biotechnology, 14, 576. doi: 10.1038/nbt0596‐576
  Wall, M. A., Socolich, M., & Ranganathan, R. (2000). The structural basis for red fluorescence in the tetrameric GFP homolog DsRed. Nature Structural Biology, 7, 1133–1138. doi: 10.1038/81992
  Wang, L., Jackson, W. C., Steinbach, P. A., & Tsien, R. Y. (2004). Evolution of new nonantibody proteins via iterative somatic hypermutation. Proceedings of the National Academy of Sciences U.S.A., 101, 16745–16749. doi: 10.1073/pnas.0407752101
  Wu, X., Simone, J., Hewgill, D., Siegel, R., Lipsky, P. E., & He, L. (2006). Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe. Cytometry Part A, 69, 477–486. doi: 10.1002/cyto.a.20300
  Yang, T. T., Cheng, L., & Kain, S. R. (1996). Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Research, 24, 4592–4593. doi: 10.1093/nar/24.22.4592
  Yu, D., Gustafson, W. C., Han, C., Lafaye, C., Noirclerc‐Savoye, M., Ge, W. P., … Shu, X. (2014). An improved monomeric infrared fluorescent protein for neuronal and tumour brain imaging. Nature Communications, 5, 3626. doi: 10.1038/ncomms4626
  Zhu, J., Musco, M. L., & Grace, M. J. (1999). Three‐color flow cytometry analysis of tricistronic expression of eBFP, eGFP, and eYFP using EMCV‐IRES linkages. Cytometry, 37, 51–59. doi: 10.1002/(SICI)1097‐0320(19990901)37:1%3c51::AID‐CYTO6%3e3.0.CO;2‐Z
PDF or HTML at Wiley Online Library