Quaternary benzo(c)phenanthridine alkaloids, new supravital DNA probes.
|Year of publication||2008|
|Type||Article in Proceedings|
|Conference||Sborník XIX. Biologických dnů|
|MU Faculty or unit|
|Field||Morphological specializations and cytology|
|Keywords||benzo(c)phenanthridine alkaloids; macarpine; DNA probe; fluorescence microscopy; flow cytometry; cell cycle|
|Description||Quaternary benzo(c)phenanthridine alkaloids (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, they are also attractive for their fluorescent properties. 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. However, most of them are restricted to use on fixed cells (e.g., ethidium bromide, propidium iodide, 7AAD, TOTO3). The most frequently used membrane permeant DNA stains are DAPI (4'6diamidino2phenylindole) 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: LDS751, DRAQ5TM. We have found that QBAs - macarpine (MA), chelirubine (CHR), sanguirubine (SR) and sanguinarine (SA) stain DNA in living cells. The best DNA staining properties showed MA. When added to intact cell suspensions, water stock solutions of MA stain nuclei of the cells upon brief (in seconds) incubation at low final concentration (0.01 to 0.001 mg/ml). Fluorescence microscopy of MA stained cells described the nuclear architecture, chromosomes and apoptotic fragments. 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 to 755 nm (i.e. fluorescence detectors FL2 to 5). Spectral characteristics of MA allow simultaneous surface immunophenotyping. The fact that MA preferentially stain nucleated cells, allows it to be used to differentiate nucleated cells from cells without a nucleus in a mixed cell population (especially blood and bone marrow cells). 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. MA staining allows differentiation of erythrocytes, reticulocytes and leukocytes in peripheral blood without the need to detect surface antigens. 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. Parameters of DNA content analysis of intact viable HL60 cells stained with MA were fully comparable with those stained with DRAQ5. Like DRAQ5, MA is quick and easy to use. Signals from RNA-MA fluorescence are low and there is no necessity for RNA digestion of living cells. Above mentioned characteristics allow multiple applications of MA providing a significant diagnostic utility.|