Ethidium bromide as a marker of mtDNA replication in living cells

Anna Maria Villa, Paola Fusi, Valentina Pastori, Giulia Amicarelli, Chiara Pozzi, Daniel Adlerstein, Silvia Maria Doglia

Research output: Contribution to journalArticle

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.

Original languageEnglish
Article number046001
JournalJournal of Biomedical Optics
Volume17
Issue number4
DOIs
Publication statusPublished - Apr 2012

Fingerprint

Ethidium
Mitochondrial DNA
markers
bromides
DNA
deoxyribonucleic acid
Cells
Fluorescence
fluorescence
Mitochondria
Confocal microscopy
Fluorescence microscopy
mitochondria
cells
Scanning
microscopy
Lasers
phenotype
scanning
Bromodeoxyuridine

Keywords

  • BrdU
  • Confocal fluorescence microscopy
  • Ethidium bromide
  • Human neuroblastoma cells
  • Ligation-mediated PCR
  • Mitochondria
  • MtDNA nucleoids mtDNA replication

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biomaterials
  • Electronic, Optical and Magnetic Materials
  • Atomic and Molecular Physics, and Optics

Cite this

Villa, A. M., Fusi, P., Pastori, V., Amicarelli, G., Pozzi, C., Adlerstein, D., & Doglia, S. M. (2012). Ethidium bromide as a marker of mtDNA replication in living cells. Journal of Biomedical Optics, 17(4), [046001]. https://doi.org/10.1117/1.JBO.17.4.046001

Ethidium bromide as a marker of mtDNA replication in living cells. / Villa, Anna Maria; Fusi, Paola; Pastori, Valentina; Amicarelli, Giulia; Pozzi, Chiara; Adlerstein, Daniel; Doglia, Silvia Maria.

In: Journal of Biomedical Optics, Vol. 17, No. 4, 046001, 04.2012.

Research output: Contribution to journalArticle

Villa, AM, Fusi, P, Pastori, V, Amicarelli, G, Pozzi, C, Adlerstein, D & Doglia, SM 2012, 'Ethidium bromide as a marker of mtDNA replication in living cells', Journal of Biomedical Optics, vol. 17, no. 4, 046001. https://doi.org/10.1117/1.JBO.17.4.046001
Villa, Anna Maria ; Fusi, Paola ; Pastori, Valentina ; Amicarelli, Giulia ; Pozzi, Chiara ; Adlerstein, Daniel ; Doglia, Silvia Maria. / Ethidium bromide as a marker of mtDNA replication in living cells. In: Journal of Biomedical Optics. 2012 ; Vol. 17, No. 4.
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