Macarpine - new supravital, red fluorescing DNA probe.
|Year of publication||2008|
|Type||Article in Proceedings|
|Conference||Book of Abstracts of XXIV International Congress ISAC.|
|MU Faculty or unit|
|Field||Morphological specializations and cytology|
|Keywords||macarpine; benzo(c)phenanthridine alkaloids; DNA probe; fluorescence microscopy; flow cytometry; cell cycle|
|Description||Introduction: Fluorescence staining of nucleic acids, especially DNA, is an important tool used in biology and diagnostics. There are a number of DNA binding fluorochromes available today, but only a few of them are cell permeable and usable for basics flow cytometres equiped with argon laser (for review see Shapiro, 2003). Macarpine is Quaternary benzo[c]phenanthridine alkaloid (QBA). QBA are naturally occurring compounds isolated from plants of Fumariaceae, Papaveraceae, Ranunculaceae and Rutaceae families. In addition to wide biological activities, they are attractive for their fluorescence (Slaninova et al., 2001, 2007). Methods: We tested fluorescent characteristics of macarpine (MA), sanguirubine (SR), chelirubine (CHR), sanguilutine (SL), chelilutine (CHL), sanguinarine (SA) and chelerythrine (CHE) upon interacting with living cells. Water stock solutions of the alkaloids (10 to 100 ug/ml) were added to intact cells and upon brief incubation the alkaloids stain the cells. Human cell lines HL60 (human promyelocytic leukemia), HeLa (human cervix adenocarcinoma) and LEP (human lung fibroblasts) and piglet blood were used in experiments. Blood cells were stained with MA in a combination with FITC conjugated anti CD45 surface marker antibody. Cells were analysed by fluorescence microscopy and by flow cytometry. Results: All tested alkaloids immediately enter living cells and MA, CHR and SA bound DNA. MA showed the best DNA staining properties. Fluorescence microscopy of MA stained cells reveals nuclear architecture and clearly defines chromosomes and apoptotic fragments in living cells. Moreover MA can rapidly report the cellular DNA content of living cells at a resolution level adequate for cell cycle analysis. QBAs were excitable by common argon lasers (488 nm) emitting at the range 575 to 755 nm (i.e. fluorescence detectors FL2to5). Spectral characteristics of MA allow simultaneous surface immunophenotyping. Conclusions: It was proven that MA, CHR and SA stain nucleic acids in living cells. They can be used as supravital fluorescent DNA probes both in fluorescence microscopy and flow cytometry including multiparameter analysis of peripheral blood. MA binds DNA stochiometrically and can report the DNA content.|