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Journal of Biomedical Optics • Open Access

Ethidium bromide as a marker of mtDNA replication in living cells
Author(s): Anna Maria Villa; Paola Fusi; Valentina Pastori; Giulia Amicarelli; Chiara Pozzi; Silvia Maria Doglia; Daniel Adlerstein

Paper Abstract

Mitochondrial DNA (mtDNA) in tumor cells was found to play an important role in maintaining the malignant phenotype. Using laser scanning confocal fluorescence microscopy (LSCFM) in a recent work, we reported a variable fluorescence intensity of ethidium bromide (EB) in mitochondria nucleoids of living carcinoma cells. Since when EB is bound to nucleic acids its fluorescence is intensified; a higher EB fluorescence intensity could reflect a higher DNA accessibility to EB, suggesting a higher mtDNA replication activity. To prove this hypothesis, in the present work we studied, by LSCFM, the EB fluorescence in mitochondria nucleoids of living neuroblastoma cells, a model system in which differentiation affects the level of mtDNA replication. A drastic decrease of fluorescence was observed after differentiation. To correlate EB fluorescence intensity to the mtDNA replication state, we evaluated the mtDNA nascent strands content by ligation-mediated real-time PCR, and we found a halved amount of replicating mtDNA molecules in differentiating cells. A similar result was obtained by BrdU incorporation. These results indicate that the low EB fluorescence of nucleoids in differentiated cells is correlated to a low content of replicating mtDNA, suggesting that EB may be used as a marker of mtDNA replication in living cells.

Paper Details

Date Published: 6 April 2012
PDF: 10 pages
J. Biomed. Opt. 17(4) 046001 doi: 10.1117/1.JBO.17.4.046001
Published in: Journal of Biomedical Optics Volume 17, Issue 4
Show Author Affiliations
Anna Maria Villa, Univ. degli Studi di Milano-Bicocca (Italy)
Paola Fusi, Univ. degli Studi di Milano-Bicocca (Italy)
Valentina Pastori, Univ. degli Studi di Milano-Bicocca (Italy)
Giulia Amicarelli, Univ. degli Studi di Milano-Bicocca (Italy)
Chiara Pozzi, Univ. degli Studi di Milano-Bicocca (Italy)
Silvia Maria Doglia, Univ. degli Studi di Milano-Bicocca (Italy)
Daniel Adlerstein, DiaSorin S.p.A. (Italy)


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