- Fluorescent probe reflects apoptosis-linked cell membrane changes
Michael J. Lander
Probes used for apoptosis detection frequently rely on plasma membrane changes that occur during the event. Fluorescently labeled annexin V, for example, interacts with phosphatidylserine that comes to the membrane surface. However, the agent requires calcium ions to function correctly and does not provide a ratiometric response.
Researchers from Université Louis Pasteur of Strasbourg, France, and from National Taras Shevchenko University and Palladin Institute of Biochemistry, both in Kiev, Ukraine, and led by Yves Mély of the former institution have developed an alternative probe that uses 4'-(diethylamino)-3-hydroxyflavone as its fluorescent reporter. It yields an emission band near 530 nm and another around 585 nm through excited-state intramolecular proton transfer.
To test the probe’s fluorescence response, the scientists stained actinomycin D-treated human lymphoid cells with it. They recorded the probe’s fluorescence spectrum with a Horiba Jobin Yvon Inc. spectrofluorometer after the cells’ excitation with 400-nm light. In apoptotic cells, the intensity ratio of the higher-wavelength band to the shorter band was 1.5 times lower than in living cells. Varying calcium concentration did not change probe response.
Next, the researchers compared the performance of the 3-hydroxyflavone-derived probe and an annexin V-based agent in flow cytometry. They used BD Biosciences cell sorters equipped with 405-nm diode and 488-nm Ar lasers and various emission filters. Based on testing with their probe, percentages of living, apoptotic and dead cells in the population were found to be 32.5, 19.5 and 48 percent, respectively. These values compared well with the 33, 18 and 49 percent measured with the other probe.
A ratiometric image of lymphatic cells shows the intensity ratio of two emission bands displayed by the probe used to stain them, indicated in the scale to the right. A lower ratio reveals a more apoptotic cell.
Both probes were used to stain cells for observation with an inverted laser scanning confocal microscope from Carl Zeiss AG. The experimental probe stained normal, apoptotic and dead cells in different colors, whereas the annexin V-based probe colored only apoptotic and necrotic cells. Because of its ratiometric response, the former also enabled assessment of the degree of apoptotic transformation. Together, the flow cytometry and microscopy results — which appeared in the Feb. 21 issue of the Journal of the American Chemical Society — point to the probe’s potential as a substitute for those based on annexin V.
Mély noted that the probe could help diagnose diseases such as cancer and help monitor the effects of drugs. He said that, in future studies, the researchers hope to modify the probes for excitation with 442-nm and 488-nm light from HeCd and Ar lasers, respectively. They also will attempt to increase photostability and residence time on the cell membrane.
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