Fluorescent Biomembrane Probe for Ratiometric Detection of Apoptosis

Herein, we developed the first ratiometric fluorescent probe for apoptosis detection. This probe incorporates selectively into the outer leaflet of the cell plasma membrane and senses the loss of the plasma membrane asymmetry occurring during the early steps of apoptosis. The high specificity to the...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2007-02, Vol.129 (7), p.2187-2193
Main Authors: Shynkar, Vasyl V, Klymchenko, Andrey S, Kunzelmann, Corinne, Duportail, Guy, Muller, Christian D, Demchenko, Alexander P, Freyssinet, Jean-Marie, Mely, Yves
Format: Article
Language:English
Subjects:
Apoptosis - physiology
Biochemistry, Molecular Biology
Cell Membrane - chemistry
Cell Membrane - metabolism
Cells, Cultured
Cellular Biology
Flavonoids - chemistry
Flavonoids - pharmacokinetics
Flow Cytometry - methods
Fluorescent Dyes - chemistry
Fluorescent Dyes - pharmacokinetics
Humans
Hydrophobic and Hydrophilic Interactions
Life Sciences
Membrane Lipids - analysis
Membrane Lipids - chemistry
Membrane Lipids - metabolism
Spectrometry, Fluorescence - methods
T-Lymphocytes - cytology
T-Lymphocytes - drug effects
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Summary:Herein, we developed the first ratiometric fluorescent probe for apoptosis detection. This probe incorporates selectively into the outer leaflet of the cell plasma membrane and senses the loss of the plasma membrane asymmetry occurring during the early steps of apoptosis. The high specificity to the plasma membranes was achieved by introduction into the probe of a membrane anchor, composed of a zwitterionic group and a long (dodecyl) hydrophobic tail. The fluorescence reporter of this probe is 4‘-(diethylamino)-3-hydroxyflavone, which exhibits excited-state intramolecular proton transfer (ESIPT), resulting in two-band emission highly sensitive to the lipid composition of the biomembranes. Fluorescence spectroscopy, flow cytometry, and microscopy measurements show that the ratio of the two emission bands of the probe changes dramatically in response to apoptosis. This response reflects the changes in the lipid composition of the outer leaflet of the cell plasma membrane because of the exposure of the anionic phospholipids from the inner leaflet at the early steps of apoptosis. Being ratiometric, the response of the new probe can be easily quantified on an absolute scale. This allows monitoring by laser scanning confocal microscopy the degree and spatial distribution of the apoptotic changes at the cell plasma membranes, a feature that can be hardly achieved with the commonly used fluorescently labeled annexin V assay.
ISSN:0002-7863
1520-5126
DOI:10.1021/ja068008h