Simultaneous Analysis of the Cyan, Green, and Yellow Fluorescent Proteins

Andrew J. Beavis1, Robert F. Kalejta1

1 Princeton University, Princeton, New Jersey
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 1.16
DOI:  10.1002/0471142956.cy0116s16
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Flow cytometric analysis of fluorescent protein expressing cells is of particular interest to researchers in many areas. The detection of fluorescent proteins in cells allows one to monitor gene expression, determine intracellular protein localization, and identify transfected cells. Wild‐type green fluorescent protein has limited utility as its spectral properties are not suitable for standard cytometers. Site‐directed mutations have produced enhanced variants with improved extinction coefficient and quantum yield with standard 488‐nm excitation. Other variants have been constructed with shifted excitation and emission maxima and high quantum yield. It is now possible to monitor multiple processes in a single cell and detect enhanced green, yellow, and cyan fluorescent proteins using a single excitation beam at 458 nm. The authors carefully describe the custom filter setup required to accomplish this and the Boolean gating logic for analysis of the various subpopulations expressing any given combination of fluorescent proteins.

PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Simultaneous Analysis of Cyan, Green, and Yellow Fluorescent Proteins
  • Support Protocol 1: Custom Cytometer Setup for Detection of Three Fluorescent Proteins
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Simultaneous Analysis of Cyan, Green, and Yellow Fluorescent Proteins

  • U‐2 OS human osteosarcoma cells (ATCC)
  • Complete medium with serum: DMEM plus 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin
  • Plasmids:
    • pEGFPN‐1, pEYFPN‐1, and pECFPN‐1 (Clontech)
    • pSP72 (Promega)
  • 1× PBS (Life Technologies or appendix 2A)
  • Hemacytometer
  • Refrigerated centrifuge
  • 0.4‐cm electrode gap cuvettes
  • BioRad gene pulser with capacitance extender
  • 10‐cm cell culture dishes
  • 12 × 75–mm (3‐ml) flow cytometry tubes
  • Flow cytometer with 458‐nm excitation and custom filter set for detection of three fluorescent proteins (e.g., FACSVantage; see protocol 2)
  • Additional reagents and equipment for trypsinizing cells ( appendix 3B)

Support Protocol 1: Custom Cytometer Setup for Detection of Three Fluorescent Proteins

  • Methanol
  • Suitable adhesive
  • Fluorescent alignment particles
  • Optical filters (Omega Optical)
  • 500‐nm LP dichroic
  • 525‐nm SP dichroic
  • 480/30 BP
  • 510/20 BP
  • 550/30 BP
  • 458 nm LP laser block
  • 458/10 nm BP
  • Lens paper
  • Dichroic mirror mounts
  • Band‐pass filter holders
  • Flow cytometer with tunable argon‐ion laser (e.g., FACSVantage)
  • Laser power meter
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Beavis, A.J. and 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.
   Cole, N.B., Smith, C.L., Sciaky, N., Teraski, M., Edidin, M., and Lippincott‐Schwartz, J. 1996. Diffusional mobility of golgi proteins in membranes of living cells. Science 273:797‐801.
   Cormack, B.P., Valdivia, R.H., and Falkow, S. 1996. FACS‐optimized mutants of the green fluorescent protein (GFP). Gene 173:33‐38.
   Cormack, B.P., Bertram, G., Egerton, M., Gow, N.A., Falkow, S., and Brown, A.J. 1997. Yeast‐enhanced green fluorescent protein (yEGFP) a reporter of gene expression in Candida albicans. Microbiology 143:303‐311.
   Cubitt, A.B., Woollenweber, L.A., and Heim, R. 1999. Understanding structure‐function relationships in the Aequorea victoria green fluorescent protein. Methods Cell Biol. 58:19‐30.
   Flach, J. and Silver, P.A. 1994. A yeast RNA‐binding protein shuttles between the nucleus and cytoplasm. Mol. Cell. Biol. 14:8399‐8407.
   Galbraith, D.W., Anderson, M.T., and Herzenberg, L.A. 1999. Flow cytometric analysis and FACS sorting of cells based on GFP accumulation. Methods Cell Biol. 58:315‐341.
   Heim, R., Prasher, D.C., and Tsien, R.Y. 1994. Wavelength mutations and posttranslational autooxidation of green fluorescent protein. Proc. Natl. Acad. Sci. U.S.A. 91:12501‐12504.
   Heim, R., Cubitt, A.B., and Tsien, R.Y. 1995. Improved green fluorescence. Nature 373:663‐664.
   Jiang, W. and Hunter, T. 1998. Analysis of cell cycle profiles in transfected cells using a membrane‐targeted GFP. BioTechniques 24:348‐354.
   Kalejta, R.F., Shenk, T., and Beavis, A.J. 1997. Use of a membrane‐localized green fluorescent protein allows simultaneous identification of transfected cells and cell cycle analysis by flow cytometry. Cytometry 29:286‐291.
   Kalejta, R.F., Brideau, A.D., Banfield, B.W., and Beavis, A.J. 1999. An integral membrane green fluorescent protein marker, Us9‐GFP, is quantitatively retained in cells during propidium iodide‐based cell cycle analysis by flow cytometry. Exp. Cell Res. 248:322‐328.
   Li, X., Zhang, G., Ngo, N., Zhao, X., Kain, S.R., and Huang, C.C. 1997. Deletions of the Aequorea victoria green fluorescent protein define the minimal domain required for fluorescence. J. Biol. Chem. 272:28545‐28549.
   Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T., and Nishimune, Y. 1997. “Green Mice” as a source of ubiquitous green cells. FEBS Lett. 407:313‐319.
   Pestov, D.G., Polonskaia, M., and Lau, L.F. 1999. Flow cytometric analysis of the cell cycle in transfected cells without cell fixation. BioTechniques 26:102‐105.
   Tsien, R.Y. 1998. The green fluorescent protein. Annu. Rev. Biochem. 67:509‐544.
   Valdivia, R.H., Hromockyj, A.E., Monack, D., Ramakrishnan, L., and Falkow, S. 1996. Applications for green fluorescent protein (GFP) in the study of host‐pathogen interactions. Gene 173:47‐52.
   Wachter, R.M., Elsliger, M.A., Kallio, K., Hanson, G.T., and Remington, S.J. 1998. Structural basis of spectral shifts in the yellow‐emission variants of green fluorescent protein. Structure 6:1267‐1277.
   Wang, S. and Hazelrigg, T. 1994. Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis. Nature 369:400‐403.
PDF or HTML at Wiley Online Library