Pj. Smith et al., Characteristics of a novel deep red/infrared fluorescent cell-permeant DNAprobe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy, CYTOMETRY, 40(4), 2000, pp. 280-291
Background: The multiparameter fluorometric analysis of intact and fixed ce
lls often requires the use of a nuclear DNA discrimination signal with spec
tral separation from visible range fluorochromes. We have developed a novel
deep red fluorescing bisalkylaminoanthraquinone, DRAQ5 (Ex lambda(max) 646
nm; E lambda(max) 681 nm; Em(lambda range) 665->800 nn), with high affinit
y for DNA and a high capacity to enter living cells. We describe here the s
pectral characteristics and applications of this synthetic compound, partic
ularly in relation to cytometric analysis of the cell cycle.
Methods: Cultured human tumor cells were examined for the ability to nuclea
r locate DRAQ5 using single and multiphoton laser scanning microscopy (LSM)
and multiparameter flow cytometry.
Results: Multiparameter flow cytometry shows that the dye can rapidly repor
t the cellular DNA content of live and fixed cells at a resolution level ad
equate for cell cycle analysis and the cycle-specific expression of cellula
r proteins (e.g., cyclin B1). The preferential excitation of DRAQ5 by laser
red lines (633/647 nm) was found to offer a means of fluorescence signal d
iscrimination by selective excitation, with greatly reduced emission overla
p with UV-excitable and visible range fluophors as compared with propidium
iodide. LSM reveals nuclear architecture and clearly defines chromosomal el
ements in live cells. DRAQ5 was found to permit multiphoton imaging of nucl
ei using a 1,047-nm emitting mode-locked YLF laser. The unusual spectral pr
operties of DRAQS also permit live cell DNA analysis using conventional 488
nm excitation and the single-photon imaging of nuclear fluorescence using
laser excitation between 488 nm and low infrared (IR; 780 nm) wavelengths.
Single and multiphoton microscopy studies revealed the ability of DRAQS to
report three-dimensional nuclear structure and location in live cells expre
ssing endoplasmic reticulum targeted-GFP, Mito-Tracker-stained mitochondria
, or a vital cell probe for free zinc (Zinquin).
Conclusion: The fluorescence excitation and emission characteristics of DRA
QS in living and fixed cells permit the incorporation of the measurement of
cellular DNA content into a variety of multiparameter cytometric analyses.
(C) 2000 Wiley Liss, Inc.