scholarly journals Β-Galactosidase-Catalyzed Fluorescent Reporter Labeling of Living Cells for Sensitive Detection of Cell Surface Antigens

2020 ◽  
Author(s):  
Katsuya Noguchi ◽  
Takashi Shimomura ◽  
Yuya Ohuchi ◽  
Munetaka Ishiyama ◽  
Masanobu Shiga ◽  
...  
Keyword(s):  
Cell Surface ◽  
Enzymatic Reaction ◽  
Wavelength Region ◽  
Living Cells ◽  
Fluorescent Dye ◽  
Surface Proteins ◽  
Quinone Methide ◽  
Fluorescence Signal ◽  
Cell Surface Antigens ◽  
Cell Surface Proteins

The ability to detect cell surface proteins using fluorescent dye-labeled antibodies is crucial for the reliable identification of many cell types. However, the different types of cell surface proteins used to identify cells are currently limited in number because they need to be expressed at high levels to exceed background cellular autofluorescence, especially in the shorter wavelength region. Herein, we report on a new method (CLAMP: quinone methide-based <u>c</u>atalyzed signa<u>l</u> <u>amp</u>lification) in which the fluorescence signal is amplified by an enzymatic reaction that strongly facilitates the detection of cell surface proteins on living cells. We used β-galactosidase as an amplification enzyme and designed a substrate for it, called MUGF, which contains a fluoromethyl group. Upon removal of the galactosyl group in MUGF by β-galactosidase labeling of the target cell surface proteins, the resulting quinone methide group-containing product was found to be both cell membrane permeable and reactive with intracellular nucleophiles, thereby providing fluorescent adducts. Using this method, we successfully detected several cell surface proteins including programmed death ligand 1 protein, which is difficult to detect using conventional fluorescent dye-labeled antibodies.

scholarly journals Β-Galactosidase-Catalyzed Fluorescent Reporter Labeling of Living Cells for Sensitive Detection of Cell Surface Antigens

2020 ◽  
Author(s):  
Katsuya Noguchi ◽  
Takashi Shimomura ◽  
Yuya Ohuchi ◽  
Munetaka Ishiyama ◽  
Masanobu Shiga ◽  
...  
Keyword(s):  
Cell Surface ◽  
Enzymatic Reaction ◽  
Wavelength Region ◽  
Living Cells ◽  
Fluorescent Dye ◽  
Surface Proteins ◽  
Quinone Methide ◽  
Fluorescence Signal ◽  
Cell Surface Antigens ◽  
Cell Surface Proteins

The ability to detect cell surface proteins using fluorescent dye-labeled antibodies is crucial for the reliable identification of many cell types. However, the different types of cell surface proteins used to identify cells are currently limited in number because they need to be expressed at high levels to exceed background cellular autofluorescence, especially in the shorter wavelength region. Herein, we report on a new method (CLAMP: quinone methide-based <u>c</u>atalyzed signa<u>l</u> <u>amp</u>lification) in which the fluorescence signal is amplified by an enzymatic reaction that strongly facilitates the detection of cell surface proteins on living cells. We used β-galactosidase as an amplification enzyme and designed a substrate for it, called MUGF, which contains a fluoromethyl group. Upon removal of the galactosyl group in MUGF by β-galactosidase labeling of the target cell surface proteins, the resulting quinone methide group-containing product was found to be both cell membrane permeable and reactive with intracellular nucleophiles, thereby providing fluorescent adducts. Using this method, we successfully detected several cell surface proteins including programmed death ligand 1 protein, which is difficult to detect using conventional fluorescent dye-labeled antibodies.

Detection and Quantification of β2AR Internalization in Living Cells Using FAP-Based Biosensor Technology

2010 ◽  
Vol 15 (6) ◽  
pp. 703-709 ◽  
Author(s):  
Gregory W. Fisher ◽  
Sally A. Adler ◽  
Margaret H. Fuhrman ◽  
Alan S. Waggoner ◽  
Marcel P. Bruchez ◽  
...  
Keyword(s):  
Cell Surface ◽  
Adrenergic Receptor ◽  
High Throughput Screening ◽  
Mammalian Cells ◽  
Living Cells ◽  
Surface Proteins ◽  
Receptor Internalization ◽  
Cell Interior ◽  
Cell Surface Proteins ◽  
Signaling Systems

Ligand-dependent receptor internalization is a feature of numerous signaling systems. In this article, the authors describe a new kind of live-cell biosensor of receptor internalization that takes advantage of fluorogen-activating protein (FAP) technology. Recombinant genes that express the human beta2 adrenergic receptor (β2AR) with FAP domains at their extracellular N-termini were transduced into mammalian cells. Exposure of the cells to membrane-impermeant fluorogens led to a strong fluorescent signal from the cell surface. Agonist-dependent translocation of the receptor from the surface to the cell interior was readily observed and quantified by fluorescence microscopy or flow cytometry in a homogeneous format without wash or separation steps. The approach described here is generalizable to other receptors and cell surface proteins and is adaptable to a variety of fluorescence-based high-throughput screening platforms.

scholarly journals Society Medal and Awards Winners for 2006

2005 ◽  
Vol 27 (4) ◽  
pp. 57-60
Keyword(s):  
Cell Surface ◽  
B Lymphocytes ◽  
Cell Types ◽  
Surface Antigens ◽  
Surface Proteins ◽  
Specific Cell ◽  
Cell Surface Antigens ◽  
Cell Surface Proteins ◽  
Extracellular Signals ◽  
Proliferation And Differentiation

Congratulations go to the following scientists who will be recipients of the Society's Awards in 2006. use antibodies against cell-surface antigens to distinguish and separate T- and B-lymphocytes and to show that antibody binding causes a redistribution and endocytosis of cell-surface proteins. He has studied the intracellular programmes and extracellular signals that control the survival, growth, proliferation and differentiation of specific cell types of the developing rodent nervous system.

scholarly journals Fluorogen-activating proteins as biosensors of cell-surface proteins in living cells

2010 ◽  
Vol 77A (8) ◽  
pp. 776-782 ◽  
Author(s):  
John Holleran ◽  
Dara Brown ◽  
Margaret H. Fuhrman ◽  
Sally A. Adler ◽  
Gregory W. Fisher ◽  
...  
Keyword(s):  
Cell Surface ◽  
Living Cells ◽  
Surface Proteins ◽  
Cell Surface Proteins

What do nanometer molecular trajectories tell us about heterogeneous cell surface domaines?

1995 ◽  
Vol 53 ◽  
pp. 786-787
Author(s):  
Watt W. Webb
Keyword(s):  
Cell Surface ◽  
Lipid Membranes ◽  
Surface Proteins ◽  
Protein Diffusion ◽  
Coated Pits ◽  
Cell Surface Proteins ◽  
Membrane Heterogeneity ◽  
Fluorescence Photobleaching Recovery ◽  
Photobleaching Recovery ◽  
Plasma Membrane Heterogeneity

Plasma membrane heterogeneity is implicit in the existence of specialized cell surface organelles which are necessary for cellular function; coated pits, post and pre-synaptic terminals, microvillae, caveolae, tight junctions, focal contacts and endothelial polarization are examples. The persistence of these discrete molecular aggregates depends on localized restraint of the constituent molecules within specific domaines in the cell surface by strong intermolecular bonds and/or anchorage to extended cytoskeleton. The observed plasticity of many of organelles and the dynamical modulation of domaines induced by cellular signaling evidence evanescent intermolecular interactions even in conspicuous aggregates. There is also strong evidence that universal restraints on the mobility of cell surface proteins persist virtually everywhere in cell surfaces, not only in the discrete organelles. Diffusion of cell surface proteins is slowed by several orders of magnitude relative to corresponding protein diffusion coefficients in isolated lipid membranes as has been determined by various ensemble average methods of measurement such as fluorescence photobleaching recovery(FPR).

Sign in / Sign up

Export Citation Format

Share Document