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 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.
Shynkar
Vasyl V
VV
Photophysique des Interactions Moléculaires, UMR 7175 CNRS, Institut Gilbert Laustriat, Faculté de Pharmacie, Université Louis Pasteur, Strasbourg 1, 74 Route du Rhin, 67401 Illkirch Cedex, France.
Klymchenko
Andrey S
AS
Kunzelmann
Corinne
C
Duportail
Guy
G
Muller
Christian D
CD
Demchenko
Alexander P
AP
Freyssinet
Jean-Marie
JM
Mely
Yves
Y
D017209:Apoptosis / Q000502:physiology*; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D002478:Cells, Cultured; D005419:Flavonoids / Q000737:chemistry* / Q000493:pharmacokinetics; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000493:pharmacokinetics; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008563:Membrane Lipids / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects
C090856:4'-diethylamino-3-hydroxyflavone; D005419:Flavonoids; D005456:Fluorescent Dyes; D008563:Membrane Lipids