Dual-color flow cytometric detection of fluorescent proteins using single-laser (488-nm) excitation

Lonnie Lybarger, Deborah Dempsey, George H. Patterson, David W. Piston, Steven R. Kain, Robert Chervenak

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

The ability to analyze independently the expression of multiple reporter gene constructs within single cells is a potentially powerful application of flow cytometry. In this paper, we explore the simultaneous detection of two variants of the reporter molecule, green fluorescent protein (GFP) that both fluoresce when excited with 488-nm light. One of these, enhanced GFP (EGFP) (excitation max. 490 nm; >90% efficiency at 488 nm), has been widely used for studies that involve flow cytometric detection of reporter gene expression. As a partner for EGFP, we employed a recently described variant termed enhanced yellow fluorescent protein (EYFP) (excitation max. 513 nm; ≃35% efficiency at 488 nm). Using 488-nm excitation, EYFP fluorescence could be readily detected following expression of the gene in murine fibroblasts and this signal was comparable in intensity to that obtained from EGYP. Importantly, we describe an optical filter configuration that permits the fluorescence signals from both proteins to be distinguished by flow cytometry, despite their similar emission maxima. This filter configuration employed a 510/20-nm bandpass filter for EGFP detection, a 550/30-nm bandpass filter for EYFP detection, and a 525-nm short-pass dichroic mirror to separate the two signals. With these filters, expression of either reporter protein could be detected, alone or in combination, within a mixed population of cells over a broad range of signal intensities.

Original languageEnglish (US)
Pages (from-to)147-152
Number of pages6
JournalCytometry
Volume31
Issue number3
DOIs
StatePublished - Mar 1 1998
Externally publishedYes

Fingerprint

Lasers
Color
Reporter Genes
Flow Cytometry
Proteins
Fluorescence
Gene Expression
Green Fluorescent Proteins
Fibroblasts
Light
Population

Keywords

  • GFP
  • Reporter gene
  • Retrovirus vector
  • YFP

ASJC Scopus subject areas

  • Hematology
  • Cell Biology
  • Pathology and Forensic Medicine
  • Biophysics
  • Endocrinology

Cite this

Dual-color flow cytometric detection of fluorescent proteins using single-laser (488-nm) excitation. / Lybarger, Lonnie; Dempsey, Deborah; Patterson, George H.; Piston, David W.; Kain, Steven R.; Chervenak, Robert.

In: Cytometry, Vol. 31, No. 3, 01.03.1998, p. 147-152.

Research output: Contribution to journalArticle

Lybarger, Lonnie ; Dempsey, Deborah ; Patterson, George H. ; Piston, David W. ; Kain, Steven R. ; Chervenak, Robert. / Dual-color flow cytometric detection of fluorescent proteins using single-laser (488-nm) excitation. In: Cytometry. 1998 ; Vol. 31, No. 3. pp. 147-152.
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