286 - Novel Fluorescent Probes for Imaging and Quantitative Analysis of Intracellular Nitric Oxide

Abstract

Nitric oxide (NO) is an important biological regulator involved in number of physiological and pathological processes. Altered NO production is implicated in various immunological, cardiovascular, neurodegenerative and inflammatory diseases. As a free radical, NO can be rapidly oxidized, thus there is relatively low concentration of NO existing in vivo. Although a variety of fluorescent sensors have been developed to detect NO, their poor photostability, short emission wavelength and low selectivity have limited the applications of these existing reactive oxygen species (ROS) and reactive nitrogen species (RNS) probes in live cells. To address this unmet need, three novel cell-permeable NO probes Nitrixyte Orange, Nitrixyte Red and Nitrixyte NIR were developed for detecting free NO in live cells. Compared to the commonly used diaminofluorescein (DAF-2), Nitrixyte probes have 1) higher selectivity toward NO than other ROS and RNS species; 2) improved sensitivity in detecting endogenous levels of NO; 3) enhanced cell permeability and 4) better photostability. Importantly, long emission wavelength of Nitrixyte Red and Nitrixyte NIR minimize autofluorescence of cells and facilitate multicolor imaging. Taking advantages of Nitrixyte probes, fluorescence imaging was successfully applied to monitor the exogenous NO in various adherent cell lines such as HeLa and CHO-K1, and to visualize the endogenously produced NO in Raw 264.7 macrophage cell line. Fluorescence microplate reader was also successfully used to quantitatively measure NO level in above cell lines. Significantly, all these novel probes were suited for monitoring intracellular NO in suspension cell lines such as T lymphocyte Jurkat cells using flow cytometer, to provide quantitative real-time measurements of NO in individual live cells. These novel fluorescent NO probes provide valuable tools for life science research and medical diagnostic applications.