Bright green-emitting ds-DNA labeling employed by dicationic monomethine cyanine dyes: Apoptosis assay and fluorescent bio-imaging

Abstract

Fluorescent based techniques are among the state-of-the-art detection methods utilized for both qualitative and quantitative investigations in biochemical and biomedical sciences. The essential methods for evaluation of apoptosis and cell cycle by measuring DNA content using flow cytometry and imaging experiments, such as fluorescent microscopy are extensively being used for staining of live and/or apoptotic cells. Among a wide diversity of commercially available fluorescent dyes, cyanine-based non-covalent labeling has been employed for several years as fluorescent probes mainly due to their capabilities for biological applications upon binding to nucleic acids.Three novel asymmetric dicationic monomethine cyanine dyes derived from 2-thiomethylbenothiazolium and 4-methylquinolinium salts were synthesized and have been tested for potential applicability in biological research as fluorescent labels for nucleic acids detection. The newly obtained cyanine probes became highly fluorescent upon addition of ds-DNA according to a well-known mechanism of this class of organic compounds. Employing flow cytometry and fluorescent microscopy, the dyes were evaluated in live-dead cells discrimination. Moreover, FACS studies on 3T3 mouse embryonic fibroblast cells verified their inability to penetrate through intact plasma membrane. Further in-depth studies on the dyes, outline their capability for estimations of cell viability and staining late apoptotic/necrotic cells.Imaging experiments by means of fluorescent microscopy, delineate the dyes' high specificity towards ds-DNA and allowed nuclear morphology imaging of non-living ethanol-fixed cells.