Green fluorescent protein: applications in cell biology

Gerdes, Hans‐Hermann; Kaether, Christoph

doi:10.1016/0014-5793(96)00586-8

Cited by 232 publications

(136 citation statements)

References 35 publications

Supporting

Mentioning

130

Contrasting

Unclassified

Order By: Relevance

“…Green fluorescent protein (GFP) and its variants have been used as fluorescent reporters in a variety of applications for monitoring dynamic processes in cells and organisms, including gene expression, protein localization, and intracellular dynamics (1,2). Optimizing the expression and brightness of GFP in mammalian cells has been of great interest.…”

mentioning

confidence: 99%

Quantification of EGFP expression on Molt‐4 T cells using calibration standards

et al. 2004

View full text Add to dashboard Cite

Background: Enhanced green fluorescent protein (EGFP) is used extensively to assess gene expression on cells; however, quantification of this expression by flow cytometry has been limited by the unavailability of calibration standards. Thus, we characterized the response of an experimental set of EGFP calibration standards to environmental changes and then quantitate the expression of EGFP, in molecules of equivalent soluble fluorochrome (MESF) units, of a transfected Molt-4 T cell line by flow cytometry. Methods: Characterization of the EGFP standards: EGFP standards were equilibrated in suspension solutions having a pH range of 5.0 -9.0, temperatures of 37-80°C, and osmolalities of 100 -600 mOsm/kg. Quantification of EGFP on cells: For transfections, Molt-4 T cells were incubated with two different concentrations (0.2 g and 0.4 g) of pEGFP-N2 vector and the EGFP expression was quantified after 48 h by flow cytometry using the EGFP standards and by the cytofluor technique using a standard curve of known EGFP solutions. Results: The fluorescence intensity of the EGFP standards increased from pH 5.0 to 9.0 and remained relatively

show abstract

mentioning

confidence: 99%

Quantification of EGFP expression on Molt‐4 T cells using calibration standards

et al. 2004

View full text Add to dashboard Cite

show abstract

“…To begin to understand the trafficking of CFTR, we constructed a jellyfish green fluorescent protein (GFP)-CFTR expression vector in which GFP was ligated to the N terminus of wild-type CFTR, and we used GFP fluorescence to localize CFTR in living and fixed cells. GFP, a 27-kDa protein from the jellyfish Aequorea victoria, has emerged as an in vivo reporter protein for studying complex biological processes such as organelle dynamics and protein trafficking (18,19). GFP generates a bright green fluorescence, is resistant to photobleaching, does not require any exogenous cofactors or substrates to fluoresce, and, when ligated to other proteins, generally does not alter fusion protein function or localization (18,20).…”

mentioning

confidence: 99%

Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells

Moyer¹,

Loffing²,

Schwiebert³

et al. 1998

Journal of Biological Chemistry

152

170

View full text Add to dashboard Cite

The mechanism by which cAMP stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride (Cl ؊ ) secretion is cell type-specific. By using Madin-Darby canine kidney (MDCK) type I epithelial cells as a model, we tested the hypothesis that cAMP stimulates Cl ؊ secretion by stimulating CFTR Cl ؊ channel trafficking from an intracellular pool to the apical plasma membrane. To this end, we generated a green fluorescent protein (GFP)-CFTR expression vector in which GFP was linked to the N terminus of CFTR. GFP did not alter CFTR function in whole cell patchclamp or planar lipid bilayer experiments. In stably transfected MDCK type I cells, GFP-CFTR localization was substratum-dependent. In cells grown on glass coverslips, GFP-CFTR was polarized to the basolateral membrane, whereas in cells grown on permeable supports, GFP-CFTR was polarized to the apical membrane. Quantitative confocal fluorescence microscopy and surface biotinylation experiments demonstrated that cAMP did not stimulate detectable GFP-CFTR translocation from an intracellular pool to the apical membrane or regulate GFP-CFTR endocytosis. Disruption of the microtubular cytoskeleton with colchicine did not affect cAMP-stimulated Cl ؊ secretion or GFP-CFTR expression in the apical membrane. We conclude that cAMP stimulates CFTR-mediated Cl ؊ secretion in MDCK type I cells by activating channels resident in the apical plasma membrane.

show abstract

“…In addition, fusion of GFP with other proteins generates labeled proteins that can monitor specific cell compartments and dynamic processes in cells. 53 Spectral variants of GFP have also been produced using mutagenesis to alter sequences in the region of the chromophore. 54 This has resulted in the blue-shifted variant (BFP), a cyan-shifted variant (CFP) and a yellow-shifted variant (YFP), all with different emission wavelengths, thus allowing simultaneous monitoring of multiple proteins within the same cell or organism.…”

Section: Fluorescence Imagingmentioning

confidence: 99%