Introduction of macarpine as a novel cell-permeant DNA dye for live cell imaging and flow cytometry sorting.
BACKGROUND
Macarpine (MA) is a quaternary benzophenanthridine plant alkaloid isolated from Macleaya microcarpa or Stylophorum lasiocarpum. Benzophenanthridine alkaloids are interesting natural products that display antiproliferative, antimicrobial, antifungal and anti-inflammatory activities, and also fluorescence properties. In a previous study, we demonstrated that thanks to its ability to interact with DNA and its spectral properties MA could be used as a supravital DNA probe for fluorescence microscopy and flow cytometry including analyses of the cell cycle. In this study, we evaluated the suitability of MA as a DNA dye for time-lapse microscopy and flow-cytometric cell sorting.


RESULTS
Living A-375 and MEF cells stained with MA were monitored by time-lapse microscopy for 24 h. Mitoses were observed at MA concentrations up to 0.5 μg/ml during the first 2-3 h. After this period of time, cells treated with MA at concentrations of 0.75 and 0.5 μg/ml underwent apoptosis. Cells cultivated with MA at concentration of 0.25 μg/ml or lower survived throughout the 24 h period. Toxicity of MA was dependent on light wavelength and frequency of image capturing. The intensity of MA fluorescence decreased during the incubation. MA concentration of 0.1 μg/ml was identified as the most suitable for live cell imaging with respect to fluorescence intensity and toxicity. MA at the concentration 10 μg/ml was used for sorting of enhanced green fluorescent protein (EGFP)-labelled neurons and fibroblasts yielding profiles similar to those obtained with DRAQ5. Contrary to DRAQ5, MA-stained cells survived in culture, and the sorted cells lost the MA signal suggesting reversible binding of the dye to the DNA.


CONCLUSIONS
The results proved that MA may readily be used for chromosomes depicting and mitosis monitoring by time-lapse microscopy. In addition, MA has shown to be a suitable probe for sorting of EGFP-labelled cells, including neurons, that survived the labelling process.


CONCLUSIONS
In consideration of the results, we highly anticipate an onward use of MA in a broad range of applications based on live cell sorting and imaging, for example, cell synchronisation and monitoring of proliferation as an important experimental and/or diagnostic utility.
Department of Biology, Faculty of Medicine, Masaryk University, Brno, 62500, Czech Republic.
Iva
I
Slaninová
Cajal Institute, IC-CSIC, Avda. Doctor Arce 37, Madrid, E-28002, Spain.
Noelia
N
López-Sánchez
Department of Biochemistry, Faculty of Medicine, Masaryk University, Brno, 62500, Czech Republic.
Kristýna
K
Šebrlová
Department of Biology, Faculty of Medicine, Masaryk University, Brno, 62500, Czech Republic.
Ondřej
O
Vymazal
Cajal Institute, IC-CSIC, Avda. Doctor Arce 37, Madrid, E-28002, Spain.
José María
JM
Frade
Department of Biochemistry, Faculty of Medicine, Masaryk University, Brno, 62500, Czech Republic.
Eva
E
Táborská
D053119:Benzophenanthridines / Q000032:analysis*; D018929:Cell Culture Techniques; D002453:Cell Cycle / Q000502:physiology*; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival; D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000032:analysis; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods
D053119:Benzophenanthridines; D005456:Fluorescent Dyes; C485184:enhanced green fluorescent protein; C000611757:macarpine; D049452:Green Fluorescent Proteins; D004247:DNA
Cell cycle; DNA dye; FACS; Macarpine; Time-lapse imaging