The use of DRAQ5 to monitor intracellular DNA in Escherichia coli by flow cytometry

Journal of Fluorescence
Filomena SilvaFernanda C Domingues

Abstract

Flow cytometry provides a rapid and high-content multiparameter analysis of individual microorganisms within a population. In the past years, several fluorescent stains were developed in order to monitor DNA content distribution and cell-cycle phases, mainly in eukaryotic cells. Recently, due to its low detection limits, several of these fluorescent stains were also applied to prokaryotic cells. In this study, the ability of a novel far-red fluorescent stain DRAQ5 in assessing intracellular DNA content distribution in Escherichia coli DH5alpha was evaluated. The results showed that a DRAQ5-labelled live E. coli suspension can be obtained by incubation of 1 x 10(6) cells/mL with 5 microM DRAQ5 in PBS buffer supplemented with EDTA (pH = 7.4) during 30 min at 37 degrees C. Flow cytometric analysis of fixed E. coli cells revealed that ethanol should be used in detriment of glutaraldehyde for DRAQ5 labelling. After the analysis of RNase and DNase digested samples, DRAQ5 was proven to be a specific DNA labelling stain. The present study demonstrates that the use of DRAQ5 as a DNA-labelling stain provides an easy assessment of intracellular DNA content and cell-cycle phases in gram-negative bacteria such as E. coli.

References

Jul 1, 1971·Journal of Bacteriology·H E Kubitschek, M L Freedman
Jun 1, 1997·Applied and Environmental Microbiology·B L RothP J Millard
Jun 30, 2000·Methods : a Companion to Methods in Enzymology·M K Winson, Hazel M Davey
Jun 20, 2003·Methods in Cell Science : an Official Journal of the Society for in Vitro Biology·Hazel M Davey
Aug 6, 2005·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Robert M MartinM Cristina Cardoso
Sep 14, 2006·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Kerenza L NjohPaul J Smith
Oct 6, 2006·Applied and Environmental Microbiology·Mónica HerreroMario Díaz
Jan 19, 2007·Clinica Chimica Acta; International Journal of Clinical Chemistry·Katrien SwertsJan Philippé
May 8, 2007·Applied and Environmental Microbiology·Covadonga QuirósMario Díaz
Apr 23, 2008·American Journal of Clinical Pathology·Robert W AllanSandra Jordan
Apr 24, 2008·BMC Microbiology·Johanna RoostaluTanel Tenson
Aug 12, 2008·Biochemistry·Saroj VelamakanniHendrik W van Veen
Sep 5, 2008·Current Protocols in Cytometry·Paul J SmithRachel J Errington
Apr 18, 2009·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Hong ZhaoZbigniew Darzynkiewicz

Citations

Nov 18, 2011·Applied Microbiology and Biotechnology·Filomena SilvaFernanda C Domingues
May 3, 2014·International Journal of Food Microbiology·Susana FerreiraFernanda C Domingues
Jun 26, 2015·Biotechnology and Bioengineering·Mauricio Mora-PaleMattheos A G Koffas
Jan 17, 2012·Biotechnology Advances·Filomena SilvaFernanda C Domingues
Jul 27, 2011·Phytomedicine : International Journal of Phytotherapy and Phytopharmacology·Filomena SilvaFernanda C Domingues
Oct 1, 2016·Applied Microbiology and Biotechnology·Benjamin BuysschaertNico Boon
Jun 8, 2014·Applied and Environmental Microbiology·Somenath BakshiJames C Weisshaar

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