Skip to main content

Analysis of Fluorescent Protein Expressing Cells by Flow Cytometry

  • Protocol
Book cover Flow Cytometry Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 263))

Abstract

The process for transfection of cells with expression and gene-trap vectors expressing fluorescent reporter proteins is described. The measurement and sorting of discrete populations of transfected cells is also described and illustrated. Of particular importance, the maintenance of stability may be important and a simple strategy to monitor this has been developed. Finally, an effective method for improving the ability to measure low-level fluorescence from autofluorescence is described.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. 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.

    Article  PubMed  CAS  Google Scholar 

  2. Lybarger, L., Dempsey, D., Patterson, G. H., Piston, D. W., Kain, S. R., and Chervenak, R. (1998) Dual-color flow cytometric detection of fluorescent proteins using single-laser (488-nm) excitation. Cytometry 31, 147–152.

    Article  PubMed  CAS  Google Scholar 

  3. Zhu, J., Musco, M. L., and 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.

    Article  PubMed  CAS  Google Scholar 

  4. Hawley, T. S., Telford, W. G., Ramezani, A., and Hawley, R. G. (2001) Four-color flow cytometric detection of retrovirally expressed red, yellow, green and cyan fluorescent proteins. Biotechniques 30, 1028–1033.

    PubMed  CAS  Google Scholar 

  5. Medico, E., Gambarotta, G., Gentile, A., Comoglio, P. M., and Soriano, P. (2001) A gene trap vector system for identifying transcriptionally responsive genes. Nat. Biotechnol. 19, 579–582.

    Article  PubMed  CAS  Google Scholar 

  6. Clontech Laboratories. (1999) Features of Living Colors™ vectors CLONTECHniques, http://www.clontech.com/gfp/GFPTable.shtml .

  7. Clontech Laboratories. (2001) Living Colors™ DsRed2 red fluorescent protein. CLONTECHniques, http://www.clontech.com/archive/JUL01UPD/pdf/LC_DsRed2.pdf.

  8. Clontech Laboratories. (2002) BD Living Colors™ HcRed red fluorescent protein. CLONTECHniques, http://www.clontech.com/archive/APR02UPD/pdf/HcRed.pdf.

  9. Sambrook, J., Fritsch, E. F, and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edit. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  10. Baird, G. S., Zacharias, D. A., and Tsien, R. Y. (2000) Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97, 11984–11989.

    Article  PubMed  CAS  Google Scholar 

  11. Zolotukhin, S., Potter, M., Hauswirth, W. W., Guy, J., and Muzyczka, N. (1996) A “humanized” green fluorescent protein cDNA adapted for high-level expression in mammalian cells. J. Virol. 70, 4646–4654.

    PubMed  CAS  Google Scholar 

  12. Stewart, C. C. and Stewart, S. J. (2003) A Software Method For Color Compensation, in Current Protocols in Cytometry (Robinson, J. P., Darzynkiewicz, Z., Dean, P., eds.), John Wiley & Sons, New York, pp. 10.15.1–10.15.12.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2004 Humana Press Inc.

About this protocol

Cite this protocol

Pruitt, S.C., Mielnicki, L.M., Stewart, C.C. (2004). Analysis of Fluorescent Protein Expressing Cells by Flow Cytometry. In: Hawley, T.S., Hawley, R.G. (eds) Flow Cytometry Protocols. Methods in Molecular Biology™, vol 263. Humana Press. https://doi.org/10.1385/1-59259-773-4:239

Download citation

  • DOI: https://doi.org/10.1385/1-59259-773-4:239

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-234-6

  • Online ISBN: 978-1-59259-773-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics