The use of DRAQ5 to monitor intracellular DNA in Escherichia coli by flow cytometry.
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.
Silva
Filomena
F
Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Avenida Infante D. Henrique, 6200-506, Covilhã, Portugal.
Lourenço
Olga
O
Pina-Vaz
Cidália
C
Rodrigues
Acácio G
AG
Queiroz
João A
JA
Domingues
Fernanda Conceição
FC
D000880:Anthraquinones / Q000378:metabolism*; D002453:Cell Cycle; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology*; D000431:Ethanol / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism*; D013379:Substrate Specificity
C520537:1,5-bis((2-(methylamino)ethyl)amino)-4,8-dihydroxyanthracene-9,10-dione; D000880:Anthraquinones; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D000431:Ethanol