| Description |
Fluorescence staining of nucleic acids, especially DNA, is an important tool used in biology and clinical diagnostics. There are a number of DNA-binding fluorochromes available today (for review see Shapiro, 2003). However, most of them are restricted to use on fixed cells (e.g., ethidium bromide, propidium iodide, 7-AAD, TOTO3). The most frequently used membrane permeant DNA stains are DAPI (4'-6-diamidino-2-phenylindole) and the bisbenzimide dyes Hoechst (33258; 33342; 34580). Both DAPI and Hoechst emit blue fluorescence under UV illumination when bound to DNA. Thus, their use is restricted to multilaser systems. Recently some cell permeable DNA dyes, excitable by argon-ion lasers (488 nm), have been introduced: LDS-751, DRAQ5 (deep red fluorescing agent 5; a synthetic antraquinone with an excitation maximum close to 700 nm, Alexis Biochemiclas) and Vybrant DyeCycleTM Green and Orange nucleic acid stains (Molecular Probes). We have found that Macarpine (MA) stain nuclei in living cells and have similar DNA staining properties as DRAQ5. MA is quaternary benzo[c]phenanthridine alkaloid (QBA). QBAs are naturally occurring compounds isolated from plants in the Fumariaceae, Papaveraceae, Ranunculaceae and Rutaceae families. In addition to having a wide range of biological activities (Zdařilová et al., 2006), they are also attractive for their fluorescent properties (Kovář et al., 1985). When added to intact cell suspensions, water stock solutions of MA stain the cells upon brief (in seconds) incubation at low final concentration (0,01-0,001 mg/ml). Human cell lines and piglet blood were used in the experiments. Blood cells were stained with MA in combination with FITC-conjugated anti-CD45 surface marker antibody. Cells were analysed by fluorescence microscopy and by flow cytometry. MA immediately entered living cells and binds to DNA. Fluorescence microscopy of MA, stained cells described the nuclear architecture and clearly described chromosomes and apoptotic fragments in living cells. Moreover MA binds to DNA stochiometrically and can rapidly represent the cellular DNA content of living cells at a resolution adequate for cell cycle analysis. MA is excitable using common argon lasers (488 nm) emitting at a range of 575 - 755 nm (i.e. fluorescence detectors FL2-5). Spectral characteristics of MA allow simultaneous surface immunophenotyping. Results, concerning the staining of blood cells, demonstrate that MA can be used for flow-cytometric cell classification on the basis of nucleic acid identification and quantification with the possibility of parallel immunophenotypisation. Furthermore, MA staining can be used in conjunction with commonly used fluorochromes that are detectable at the FL1 detector (FITC, GFP) of either standard or 2-laser systems. We concluded that MA can be used as supravital fluorescent DNA probe, both in fluorescence microscopy and flow cytometry, including multiparameter analysis of peripheral blood. MA binds DNA stochiometrically and can provide information on DNA content.
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