Rapid generation and flow cytometric analysis of stable GFP-expressing cells

Lonnie Lybarger, Deborah Dempsey, Karl J. Franek, Robert Chervenak

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Expression of green fluorescent protein (GFP) represents a unique method for the fluorescent labeling of viable mammalian cells, with many potential applications. The studies detailed in this report examine the detection of GFP expression in murine cells using flow cytometry. Direct comparison of NIH 3T3 cells transiently expressing GFP or GFPS65T, a mutant form of GFP, showed that GFPS65T fluorescence (using 488 nm excitation) was detected more readily than fluorescence from wildtype GFP. Efficient generation of cell lines that stably express GFPS65T was achieved using a plasmid vector that encoded a hygromycin phosphotransferase/GFPS65T fusion protein. Flow cytometric detection of NIH 3T3 cells expressing this fusion protein was improved using a 510/20 band pass filter instead of the standard filter setup for fluorescein detection. Additionally, staining the surface of these cells with phycoerythrin, RED 670, or allophycocyanin did not interfere with the detection of GFPS65T fluorescence, indicating that multiparameter analyses using GFPS65T fluorescence are possible. Importantly, we also observed that GFPS65T expression could be detected in NIH 3T3, BW5147, or freshly cultured Thy1(lo) CD3- murine bone marrow cells transduced with a retroviral vector encoding the fusion protein, suggesting that the potential applications of this system may be quite broad.

Original languageEnglish (US)
Pages (from-to)211-220
Number of pages10
JournalCytometry
Volume25
Issue number3
DOIs
StatePublished - Nov 1 1996
Externally publishedYes

Fingerprint

Green Fluorescent Proteins
Fluorescence
NIH 3T3 Cells
hygromycin-B kinase
Phycoerythrin
Proteins
Cell Fusion
Fluorescein
Bone Marrow Cells
Flow Cytometry
Plasmids
Staining and Labeling
Cell Line

Keywords

  • Gene expression
  • Green fluorescent protein
  • Multiparameter analysis
  • Retrovirus

ASJC Scopus subject areas

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

Cite this

Rapid generation and flow cytometric analysis of stable GFP-expressing cells. / Lybarger, Lonnie; Dempsey, Deborah; Franek, Karl J.; Chervenak, Robert.

In: Cytometry, Vol. 25, No. 3, 01.11.1996, p. 211-220.

Research output: Contribution to journalArticle

Lybarger, Lonnie ; Dempsey, Deborah ; Franek, Karl J. ; Chervenak, Robert. / Rapid generation and flow cytometric analysis of stable GFP-expressing cells. In: Cytometry. 1996 ; Vol. 25, No. 3. pp. 211-220.
@article{c80ac7891b1f462abde6f225762796d7,
title = "Rapid generation and flow cytometric analysis of stable GFP-expressing cells",
abstract = "Expression of green fluorescent protein (GFP) represents a unique method for the fluorescent labeling of viable mammalian cells, with many potential applications. The studies detailed in this report examine the detection of GFP expression in murine cells using flow cytometry. Direct comparison of NIH 3T3 cells transiently expressing GFP or GFPS65T, a mutant form of GFP, showed that GFPS65T fluorescence (using 488 nm excitation) was detected more readily than fluorescence from wildtype GFP. Efficient generation of cell lines that stably express GFPS65T was achieved using a plasmid vector that encoded a hygromycin phosphotransferase/GFPS65T fusion protein. Flow cytometric detection of NIH 3T3 cells expressing this fusion protein was improved using a 510/20 band pass filter instead of the standard filter setup for fluorescein detection. Additionally, staining the surface of these cells with phycoerythrin, RED 670, or allophycocyanin did not interfere with the detection of GFPS65T fluorescence, indicating that multiparameter analyses using GFPS65T fluorescence are possible. Importantly, we also observed that GFPS65T expression could be detected in NIH 3T3, BW5147, or freshly cultured Thy1(lo) CD3- murine bone marrow cells transduced with a retroviral vector encoding the fusion protein, suggesting that the potential applications of this system may be quite broad.",
keywords = "Gene expression, Green fluorescent protein, Multiparameter analysis, Retrovirus",
author = "Lonnie Lybarger and Deborah Dempsey and Franek, {Karl J.} and Robert Chervenak",
year = "1996",
month = "11",
day = "1",
doi = "10.1002/(SICI)1097-0320(19961101)25:3<211::AID-CYTO2>3.0.CO;2-I",
language = "English (US)",
volume = "25",
pages = "211--220",
journal = "Cytometry",
issn = "0196-4763",
publisher = "John Wiley and Sons Inc.",
number = "3",

}

TY - JOUR

T1 - Rapid generation and flow cytometric analysis of stable GFP-expressing cells

AU - Lybarger, Lonnie

AU - Dempsey, Deborah

AU - Franek, Karl J.

AU - Chervenak, Robert

PY - 1996/11/1

Y1 - 1996/11/1

N2 - Expression of green fluorescent protein (GFP) represents a unique method for the fluorescent labeling of viable mammalian cells, with many potential applications. The studies detailed in this report examine the detection of GFP expression in murine cells using flow cytometry. Direct comparison of NIH 3T3 cells transiently expressing GFP or GFPS65T, a mutant form of GFP, showed that GFPS65T fluorescence (using 488 nm excitation) was detected more readily than fluorescence from wildtype GFP. Efficient generation of cell lines that stably express GFPS65T was achieved using a plasmid vector that encoded a hygromycin phosphotransferase/GFPS65T fusion protein. Flow cytometric detection of NIH 3T3 cells expressing this fusion protein was improved using a 510/20 band pass filter instead of the standard filter setup for fluorescein detection. Additionally, staining the surface of these cells with phycoerythrin, RED 670, or allophycocyanin did not interfere with the detection of GFPS65T fluorescence, indicating that multiparameter analyses using GFPS65T fluorescence are possible. Importantly, we also observed that GFPS65T expression could be detected in NIH 3T3, BW5147, or freshly cultured Thy1(lo) CD3- murine bone marrow cells transduced with a retroviral vector encoding the fusion protein, suggesting that the potential applications of this system may be quite broad.

AB - Expression of green fluorescent protein (GFP) represents a unique method for the fluorescent labeling of viable mammalian cells, with many potential applications. The studies detailed in this report examine the detection of GFP expression in murine cells using flow cytometry. Direct comparison of NIH 3T3 cells transiently expressing GFP or GFPS65T, a mutant form of GFP, showed that GFPS65T fluorescence (using 488 nm excitation) was detected more readily than fluorescence from wildtype GFP. Efficient generation of cell lines that stably express GFPS65T was achieved using a plasmid vector that encoded a hygromycin phosphotransferase/GFPS65T fusion protein. Flow cytometric detection of NIH 3T3 cells expressing this fusion protein was improved using a 510/20 band pass filter instead of the standard filter setup for fluorescein detection. Additionally, staining the surface of these cells with phycoerythrin, RED 670, or allophycocyanin did not interfere with the detection of GFPS65T fluorescence, indicating that multiparameter analyses using GFPS65T fluorescence are possible. Importantly, we also observed that GFPS65T expression could be detected in NIH 3T3, BW5147, or freshly cultured Thy1(lo) CD3- murine bone marrow cells transduced with a retroviral vector encoding the fusion protein, suggesting that the potential applications of this system may be quite broad.

KW - Gene expression

KW - Green fluorescent protein

KW - Multiparameter analysis

KW - Retrovirus

UR - http://www.scopus.com/inward/record.url?scp=0029952109&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029952109&partnerID=8YFLogxK

U2 - 10.1002/(SICI)1097-0320(19961101)25:3<211::AID-CYTO2>3.0.CO;2-I

DO - 10.1002/(SICI)1097-0320(19961101)25:3<211::AID-CYTO2>3.0.CO;2-I

M3 - Article

VL - 25

SP - 211

EP - 220

JO - Cytometry

JF - Cytometry

SN - 0196-4763

IS - 3

ER -