Journal of Immunological Methods 229 Ž1999.

131–139
www.elsevier.nlrlocaterjim

A novel cell permeant and far red-fluorescing DNA probe,

DRAQ5, for blood cell discrimination by flow cytometry
a,),1
Paul J. Smith , Marie Wiltshire a , Sharon Davies a , Laurence H. Patterson b,
Terence Hoy c
a
Department of Pathology, UniÕersity of Wales, College of Medicine, Heath Park, Cardiff, CF4 4XN, UK
b
Department of Pharmaceutical Sciences, De Montfort UniÕersity, Leicester, LE1 9BH, UK
c
Department of Haematology, UniÕersity of Wales, College of Medicine, Heath Park, Cardiff, CF4 4XN, UK
Received 2 October 1998; received in revised form 24 March 1999; accepted 2 August 1999

Abstract

The deep red fluorescing agent ŽDRAQ5. is a synthetic anthraquinone with a high affinity for DNA and a high capacity
to rapidly enter living cells or stain fixed cells. DRAQ5 is optimally excited by red-light emitting sources and yields a deep
red emission spectrum which extends into the low infra-red. DRAQ5 shows excitation at sub-optimal wavelengths including
the 488 nm line and the multi-line UV wavelengths emitted by argon-ion lasers. Single beam Ž488 nm. flow cytometry has
been used to demonstrate the utility of DRAQ5-nuclear DNA fluorescence as a discriminating parameter for human
leucocytes and lymphoma cells, in combination with fluorochrome-labelled antibodies for the detection of surface antigens
and subpopulation recognition. DRAQ5 fluorescence was found to reflect cellular DNA content as evidenced by cell cycle
distribution profiles for asynchronous and cell cycle-perturbed populations. Importantly, DRAQ5 can be used in combination
with FITC and RPE-labelled antibodies, without the need for fluorescence compensation. q 1999 Elsevier Science B.V. All
rights reserved.

Keywords: DNA; Fluorochrome; Cytometry; Leucocyte; Vital cell stain

1. Introduction many analytical methods employing flow and laser

The fluorometric detection of cellular DNA or
chromosomal material is an important component of
AbbreÕiations: FITC, fluorescein isothiocyanate; FSC, For-
ward light scatter; RPE, R-phycoerythrin; PBS, Phosphate buffered
saline; PMT, Photomultiplier tube; BSA, Bovine serum albumin;
UV, Ultra violet; DNA, Deoxyribonucleic acid; RNA, Ribonucleic
acid
)
Corresponding author. Tel.: q44-1222-742730; fax: q44-
1222-744276; e-mail: smithpj2@cf.ac.uk
1
Correspondence regarding supplies of DRAQ5 should be sent
to PJS.
scanning cytometry. The discrimination of intact nu-
cleated cells in complex intact populations is a fun-
damental aspect of multiparameter analytical proce-
dures. Such nuclear detection may be required for a
variety of reasons including the enhancement of
assay specificity and simplicity, exclusion of cell
doublets and debris, and the maintenance of cellular
integrity for epitope presentation or the monitoring
of cellular biochemistry. Conversely, the exclusion
of nucleated cells is essential in assays involving
mature red blood cells and platelets. Discrimination
can be on the basis of nuclear staining potential or

0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 0 2 2 - 1 7 5 9 Ž 9 9 . 0 0 1 1 6 - 7
132 P.J. Smith et al.r Journal of Immunological Methods 229 (1999) 131–139
DNA content, the latter being required when cell
cycle dependence or perturbation needs to be evalu-
ated. There are a number of DNA-binding fluo-
rochromes available Žfor reviews see, Latt and Lan-
glois, 1990; Waggoner, 1990; Darzynkiewicz and
Kapuscinski, 1990. which cover the UV and visible
regions of the spectrum with convenient Ex l maxr
Em l max values such as: Hoechst 33258 Ž346 nmr460
nm., DAPI Ž350 nmr470 nm., acridine orange Ž502
nmr526 nm., ethidium bromide Ž510 nmr595., pro-
pidium iodide Ž536 nmr617 nm., and the recently
described DNA-intercalating cyanine fluorochromes
TOTO 1 Ž514 nmr533 nm. and TOTO 3 Ž642
nmr661 nm..
Conventionally, propidium iodide is used to iden-
tify cellular DNA content but cells need to be fixed
and RNase digested. Aminoactinomycin D Ž7-AAD.,
selective for GC-rich DNA regions, has also been
used to discriminate cellular DNA content by multi-
parameter flow cytometry ŽRabinovitch et al., 1986..
Although 7-AAD can be excited by the argon-ion
laser, emitting beyond a wavelength of 610 nm, it
appears to be generally excluded from intact viable
cells. The bisbenzimide dyes, Hoechst 33258 and
Hoechst 33342, are cell-permeant, minor groove
binding DNA stains that fluoresce bright blue upon
binding to DNA. Hoechst 33342 has higher mem-
brane permeability than Hoechst 33258, but both
dyes require UV excitation ŽSmith et al., 1985; Wat-
son et al., 1985..
Some SYTO nucleic acid stains ŽMolecular
Probes, Eugene, OR; Frey et al., 1995. are character-
istically able to penetrate intact live cells. Although
SYTO stains can be used as visible light excitable
dyes for labelling DNA and RNA they do not act
exclusively as nuclear stains in live cells and should
therefore not be equated with compounds such as
Hoechst 33258 or Hoechst 33342. Members of the
SYTO series have different spectral properties. In
particular some have excitation and emission spectra
similar to those of fluorescein and thus cannot be
used with FITC-labelled antibodies. SYTO-16 has
been used as an apoptotic discriminator ŽFrey, 1995..
A red fluorescent derivative ŽSYTO 17. has excita-
tionremission maxima ; 621r634 nm for the
dye–nucleic acid complex, which results in a signifi-
cant overlap with the excitationremission maxima of
the commonly used Texas Red fluorophor. LDS-751
is a cell-permeant nucleic acid stain that shows
fluorescence enhancement on binding to DNA and
RNA. LDS-751 has been used to discriminate intact
nucleated cells from non-nucleated cells and despite
a peak excitation at ; 543 nm on dsDNA, the
fluorophor can be excited by the argon ion laser at
488 nm, exhibiting a far-red emission maximum
ŽTerstappen et al., 1988, 1989.. LDS-751 binds to
double-stranded DNA and RNA and there has yet to
be any demonstration of its utility in discriminating
cellular DNA content at the resolution required for
cell cycle analysis although it can give adequate
differentiation of apoptotic from non-apoptotic cells
in a model thymocyte system ŽFrey, 1995..
Each of the agents discussed above has one or
more deficiencies which restrict their use with intact
cells and conventional argon ion laser-equipped flow
cytometers as used in blood cell analysis. Accord-
ingly, agents may lack cell permeant properties, have
emission spectra which overlap with those of other
relevant fluorophors, lack sufficient DNA specificity
for critical DNA content discrimination or require
UV-excitation. Here we describe the characteristics
of the new agent, DRAQ5, with properties which
circumvent the shortcoming of other agents ŽSmith
and Patterson, 1998.. DRAQ5 is a 1,5-bisw2-Žmeth-
ylamino .ethylxamino4 -4,8-dihydroxy anthracene-
9,10-dione, designed with the aid of molecular mod-
elling to inform the effects of side-chain composition
on the DNA binding properties which would alter
the intracellular fluorescence characteristics of cell
permeant anthraquinones ŽPatterson, 1989; Smith et
al., 1997a,b.. DRAQ5 achieves nuclear discrimina-
tion by its high affinity mode of DNA intercalation
and the nature of its modelled base-pair specificity.
Excitation at a wavelength of 647 nm, close to the
Ex l max , produces a fluorescence spectrum extending
from 665 nm out to beyond 780 nm wavelengths.
Thus the emission spectrum is beyond that of fluo-
rescein, phycoeryrthrin, Texas Red and Cy 3. At
high DNA:DRAQ5 ratios a spectral shift contributes
to a partial separation of the DRAQ5-DNA emission
spectrum from that of Cy 5.
Preliminary studies revealed that DRAQ5 could
be sub-optimally activated by the 488 nm line of an
argon ion laser. Here we describe the ability of this
novel far redrinfra-red fluorescing agent to stain the
DNA, of viable human cells, in comparison with
 P.J. Smith et al.r Journal of Immunological Methods 229 (1999) 131–139
LDS-751, and demonstrate its application to the
rapid analysis of whole and isolated blood prepara-
tions.
2. Materials and methods
2.1. Cell culture and treatments
The SUD4 human B cell follicular lymphoma cell
line Žkindly provided by Dr. F. Cotter, Institute of
Child Health, London. was grown as suspension
cultures in RPMI medium with 10% foetal calf
serum, 1 mM glutamine and antibiotics and incu-
bated at 378C in an atmosphere of 5% CO 2 in air.
SUD4 is a mutant of the tumour suppressor gene
TP53 . has reduced commitment to apoptosis in re-
sponse to DNA damaging agents. For flow cytome-
try experiments, asynchronously growing suspension
cultures were diluted to 2.5–4 = 10 5 cellsrml at 2 h
prior to drug treatment. Cell cycle-perturbed popula-
tions were obtained by treating SUD4 cells with the
anticancer drug etoposide ŽVP-16; Vepeside; Bristol
Myers. at 0.25 mM for 18 h. VP-16 acts as a DNA
topoisomerase II poison and results in the arrest of
cells in late S phase and G2 of the cell cycle.
DRAQ5 was synthesised as described previously
ŽSmith and Patterson, 1998. and prepared as an
aqueous 10 mM solution stored at 48C. Human blood
was obtained using routine venepuncture of a healthy
donor and samples manipulated using standard
haematological procedures for the isolation of
mononuclear blood cells and surface antigen recogni-
tion using antibody panels. Cells were treated with
DRAQ5 as described and analysed by flow cytome-
try without washing. Suspension cultures were anal-
ysed by flow cytometry without washing.
2.2. Preparation of cells for flow cytometry
2.2.1. Preparation 1
SUD4 cells were resuspended in analysis buffer
ŽPBS q 0.5% BSA q 0.02% sodium azide.. Parallel
analysis of samples using conventional ethidium bro-
mide staining of RNase A-digested permeabilised
cells was used to determine cell cycle phase distribu-
tion for asynchronous cultures and for late cell
133
cycle-arrested populations ŽWatson et al., 1987; Ep-
stein et al., 1988..
2.2.2. Preparation 2
As for Preparation 1 but processed for surface
antigen analysis using directly labelled antibodies:
anti-CD45-FITC Ždetected by the FL1 photomulti-
plier; i.e., FL1 PMT., anti-CD19-PE ŽFL2 PMT..
2.2.3. Preparation 3
Ficoll-separated viable mononuclear blood cells
were prepared from a normal donor. Sample ob-
tained by routine venepuncture and resuspended in
analysis buffer.
2.2.4. Preparation 4
As for preparation 3 but processed for surface
antigen analysis using anti-CD45-FITC ŽFL1 PMT..
2.2.5. Preparation 5
Whole blood from a normal donor was processed
for surface antigen analysis, analysed as viable cells
using anti-CD45-FITC positively as the FL1 parame-
ter master trigger to exclude RBCs.
2.2.6. Preparation 6
As for preparation 5 but after processing for
surface antigen analysis, cells were fixed and RBCs
lysed in FACSLysee; Becton Dickinson, Cowley,
UK. and re-suspended in analysis buffer. The FSC
parameter was used as the master trigger.
2.2.7. Preparation for fluorochrome comparisons
Cells in full culture medium were stained for: Ži.
10 min with ethidium bromide Ž50 mgrml. plus
0.125% Triton X-100 and 0.5 mgrml ribonuclease
A, Žii. exposed to LDS-751 Ž0.2 mgrml. ŽExciton,
Dayton, OH. for 30 min at room temperature, or Žiii.
exposed to 20 mM DRAQ5 for 30 min at room
temperature.
2.3. Flow cytometry
DRAQ5 can be activated at multiline UV, or 488
nm, or 514 nm, or 568 nm, or 633 nm, or 647 nm
Žwith increasing efficiency as the wavelengths in-
crease.. Here we have used two cytometer systems.
134 P.J. Smith et al.r Journal of Immunological Methods 229 (1999) 131–139
First a single beam low power laser excitation
provided by the FACScan system ŽBecton Dickin-
son, Cowley, UK. which incorporates an argon ion
laser Žmax 15 mW output., tuned to the 488-nm line.
Forward light scatter, 908 light scatter and fluores-
cence emissions ŽFL parameters. were collected for
1 = 10 4 cells using the forward light scatter ŽFSC.
parameter, where appropriate, as the master signal.
The standard analysis optics provided the FL1
Žgreen., FL2 Žorange., and FL3 Žred;) 650 nm.
PMT parameters with pulse analysis performed on
the FL3 originating signals. Pulse height measure-
ments were displayed for FL1 and FL2 parameters,
with pulse area for the FL3 parameter.
Second, to compare fluorochrome staining charac-
teristics cells were analysed using a FACS Vantage
cell sorter ŽBecton Dickinson, Cowley, UK. incorpo-
rating a Coherent Enterprise II laser simultaneously
emitting at multiline UV Ž350–360 nm range. and
488 nm wavelengths with the beams made non-co-
linear using dichroic separators. Forward light scat-
ter, 908 light scatter and fluorescence emissions were
collected for 1 = 10 4 cells using the forward light
scatter parameter as the master signal from the pri-
mary 488 nm beam, while side scatter was collected
through a 488r10 nm band-pass filter. The analysis
optics were: Ži. primary beam-originating signals
analysed at FL1 ŽFITC filter; barrier filter of 530r30
nm. after transmission at SP610 and SP560 dichroics,
or at FL2 Žbarrier filters of 585r42 nm. after trans-
mission at SP610 and reflection at SP560 dichroics,
or at FL3 Žbarrier filter of LP715 nm. after reflection
at a SP610 dichroic; Žii. delayed beam-originating
signals analysed at FL4 Žbarrier filter of LP695 nm.
or at FL5 Žbarrier filter of DF424r44 nm. after
transmission or reflection at a LP640 dichroic, re-
spectively. Forward and 908light scatter were anal-
ysed to exclude any cell debris. All parameters were
analysed as pulse height using CellQuest software
ŽBecton Dickinson..
3. Results
We have sought to demonstrate the utility of
DRAQ5 in a single beam cytometer Ži.e., a FAC-
Scane.. Fig. 1a–d shows typical results demonstrat-
ing the ability of DRAQ5 to identify nucleated cells
in complex populations. Fig. 1a shows the detection
of cell cycle distribution vs. cell surface antigen
expression for intact cells. Fig. 1b shows the discrim-
ination of subsets according to staining potential
while Fig. 1c and d demonstrate the application of
DRAQ5 in detecting nucleated cells in whole blood
and lysed blood. Factors relating to the ability of
DRAQ5 to stain nuclei are analysed in Table 1.
Using viable cultured asynchronous lymphoma
cells ŽPreparation 1. DRAQ5 rapidly stained cells in
a reproducible manner and generated fluorescence
distinct from the low autofluorescence background.
The large standard deviation values derive from the
expected spread of cells throughout the cell cycle
Žanalyses given in footnote to Table 1.. The mean
value reflects the cellular DNA content as evidenced
by the 1.7-fold increase for G2 arrested populations.
The processing of cells for surface antigen analysis
ŽPreparation 2. does not affect the above character-
istics. The isolation of intact mononuclear blood
cells ŽPreparations 3 and 4. yielded samples which
could be stained within a convenient cell density
range and be processed for surface antigen analysis.
In Preparations 3 and 4 we consistently observed an
enhanced staining potential of monocytes vs. lym-
phocytes Ž1.14–1.4 fold.. It is likely that enhanced
staining relates to the accessibility of DNA binding
sites, these differing according to cell type as has
been observed for the Hoechst dyes ŽSmith et al.,
1985.. We suggest that it is likely that optimal
staining conditions have not been achieved and
should be investigated according to the cell type
under investigation. However, such differences in
viable cell staining potential, if maximised, may be
used as a means of subpopulation discrimination.
The results for whole blood suggest that nucleated
cells Žincluding granulocytes. can be stained to a
similar degree in the presence ŽPreparation 5. of red
blood cells ŽRBCs. or following RBC lysis and mild
fixation ŽPreparation 6..
Fig. 2 summarises the DRAQ5 concentration-de-
pendent differences in DRAQ5 staining for viable
cell populations obtained using Preparation methods
1 and 2. The populations show similar titration curves
with saturation occurring in a manner which reflects
relative DNA content Žfor a given cell type, e.g.,
 P.J. Smith et al.r Journal of Immunological Methods 229 (1999) 131–139 135

Fig. 1. Single beam flow cytometric analysis ŽFACScan. of DRAQ5 fluorescence ŽFL3-area. vs. antibody fluorescence ŽFL2-height
monitoring phycoerythrin-labelled anti-CD19 or FL1-height monitoring FITC-labelled anti-CD45. for cultured and blood-derived human
cells. Cell suspensions Ž2.5 = 10 5 rml. were maintained in phosphate buffered saline containing 1% bovine serum albumin. Human blood
mononuclear cell subpopulations, obtained using standard Ficoll gradient separation, were identified and gated according to their forward-
and side-light scatter characteristics. Doublets were excluded by pulse analysis gating on normal FL3-area vs. FL3-width parameter values.
Panel a: cultured asynchronous SUD4 lymphoma cells. Panel b: blood mononuclear cell subpopulations, obtained using standard Ficoll
gradient separation. Panel c: whole blood Žtriggered on CD45q events.. Panel d: lysed whole blood. Arrowed subpopulations: G1, S and
G2rM represent cell cycle phases; L, lymphocytes; M, monocytes; G, granulocytes; N, nuclei lacking plasma membranes.

SUD4. or nuclear staining potential Že.g., lympho- DNA content, and to demonstrate a far-red fluores-
cytes vs. monocytes. at concentrations of ) 10 mM. cence signature. Fig. 3 shows the intensity distribu-
We have compared the ability of DRAQ5 and tions for cells stained with DRAQ5 or LDS-751
LDS-751 to stain viable cells, provide resolution of compared with that achieved using propidium iodide
136 P.J. Smith et al.r Journal of Immunological Methods 229 (1999) 131–139

Table 1
FACScane flow cytometric analysis of DRAQ5-labelled human cells
Cell preparation and DRAQ5 exposure period Žmin. Mean fluorescence intensity Ž"SD. of gated populationa
SUD4 cells Lymphocytes Monocytes Granulocytes
b
Preparation 1: Õiable cultured cells
0 1.3 " 6.8
5 Ž1 st sample. 499.4 " 143.3
5 Ž2nd sample. 532.4 " 148.3
120 645.1 " 177.7
0 Ž0.25 mM VP-16. 6.0 " 16.6
5 Ž0.25 mM VP-16. 891.8 " 126.4

Preparation 2: Õiable cultured cells, surface antigen analysis c

0 5.2 " 13.8
5 564.3 " 147.2
0 Ž0.25 mM VP-16. 13.4 " 21.6
5 Ž0.25 mM VP-16. 902.7 " 117.1

Preparation 3: Ficoll gradient-isolated Õiable mononuclear blood cells d

0 0.8 " 4.6 1.7 " 5.7
5 227.4 " 24.5 302.9 " 25.4
5 Ž4.8 = 10 5 rml. 215.9 " 26.3 301.8 " 24.3
5 Ž7.5 = 10 5 rml. 234.1 " 27.0 311.0 " 30.9
120 299.1 " 23.9 342.2 " 22.4

Preparation 4: Preparation 3 plus surface antigen analysis e

0 0.0 " 0.1 0.0 " 0.1
5 261.6 " 26.3 317.9 " 27.3

Preparation 5: Whole blood, Õiable cells surface antigen analysis f

0 0.0 " 0.2 0.1 " 1.1 0.1 " 2.1
5 257.3 " 35.0 274.6 " 41.0 248.4 " 35.9

Preparation 6: Preparation 5 but cells lysed and fixed g

0 0.0 " 0.3 0.0 " 0.9 0.1 " 3.0
5 228.5 " 28.5 240.2 " 28.1 254.3 " 31.1
a
Fluorescence detected on FL3 and analysed as pulse area parameter for cell populations gated on the relevant forward-scatter ŽFSC. and
side-scatter ŽSSC. characteristics. All cell preparations analysed at 2.5 = 10 5rml, unless otherwise indicated, in phosphate buffered saline
plus 1% BSA Ži.e., analysis buffer. without Ž0 min. or with Ž5 or 120 min exposure. 20 mM DRAQ5.
b
Preparation 1: Cell cycle analysis yielded G1 s 34.9%, S phase s 48.0%, G2rMs 17.1% for asynchronous cultures, and G1 s 0.6%, S
phase s 55.3%, G2rMs 44.0% for late cell cycle-arrested cells. Quantitative analysis of DNA content of G1 ŽSUD4.: G1 Žnormal diploid
lymphocytes. gave a ratio of 1.083.
c
Preparation 2: Surface antigen analysis using anti-CD45-FITC and anti-CD19-PE.
d
Preparation 3: Ratio lymphocytes:monocytess 11.5:1
e
Preparation 4: Surface antigen analysis using anti-CD45-FITC.
f
Preparation 5: Gated populations of 25.5% lymphocytes, 13.3% monocytes, 61.2% granulocytes.
g
Preparation 6: Gated populations of 43.7% lymphocytes, 9.7% monocytes, 46.6% granulocytes.

stained cells. DRAQ5 provides resolution of G1, S
phase and G2 DNA contents of intact cells compara-
ble with that obtained using propidium iodide in
fixed cells. There was no resolution of DNA content
using LDS-751.
The fluorescence characteristics of viable SUD4
cells exposed to 0.2 mgrml LDS-751 or 20 mM
DRAQ were compared for two different excitation
conditions. First, excitation at 488 nm gave mean
fluorescence values of y1.6 " 3.5 and 1.8 " 1.1 for
 P.J. Smith et al.r Journal of Immunological Methods 229 (1999) 131–139
FL1 parameter Ž530r30 nm., 70.7 " 5.3 and 13.8 "
18.3 for FL2 parameter Ž585r42 nm., 567.4 " 59.4
and 346.3 " 90.5 for FL3 parameter Ž) 715 nm.
with LDS-751 and DRAQ, respectively. Second, ex-
citation using multiline UV Ž350–360 nm. gave mean
fluorescence values of 238.7 " 27.6 and 299.6 " 67.6
for FL4 parameter Ž) 695 nm., and 0.3 " 0.1 and
0.2 " 0.1 for FL5 parameter Ž424r44 nm. with
LDS-751 and DRAQ, respectively. The results show
that both LDS-751 and DRAQ5 can be excited by
488 nm wavelength or multiline UV light with simi-
lar far-red emission intensities. There were no signif-
icant signals, beyond the autofluorescence back-
ground, obtained from DRAQ5 monitored by the
FL1 and FL5 detectors. For blue line excitation there
are significant levels of LDS-751-associated cellular
fluorescence monitored by the FL2 parameter
Ž585r42 nm. while the ‘‘breakthrough’’ signal in
this region is 5-fold lower for DRAQ5 and not
Fig. 2. Flow cytometric quantification ŽFACScan. of fluorescence
intensity of cultured and blood-derived human cells exposed to
DRAQ5 at room temperature for 5 min. Cell suspensions Ž2.5=
10 5 rml. were maintained in phosphate buffered saline containing
1% bovine serum albumin. Human blood mononuclear cell sub-
populations, obtained using a standard Ficoll gradient separation,
were identified and gated according to their forward- and side-light
scatter characteristics. Data are mean values Ž"SD. and represent
results from a typical experiment. Symbols: open circle, cultured
asynchronous SUD4 lymphoma cells; open square, SUD4 cells
exposed to 0.25 mM VP-16 for 18 h to arrest cells in S phase and
G2 of the cell cycle; closed circle, lymphocytes; closed square,
monocytes.
137
Fig. 3. Typical DNA content distributions ŽFL3., analysed by flow
cytometry ŽFACS Vantage. of LDS-751 Žbroken line. or DRAQ5
Žbold line. stained viable SUD4 cells. The fine line shows the
overlayed distribution, obtained at lower FL3 amplifier settings
for ethanol-fixed propidium-iodide stained cells.
significantly greater than the autofluorescence back-
ground.
4. Discussion
DRAQ5 can be considered as a new fluorochrome
for the detection of nucleated cells in fluorescence-
based systems. Potential applications could exploit
the far redrinfra-red fluorescence emission spectrum
andror its capacity to bind to DNA, while there
appears to be only a minimal signal Ž- 10%. derived
from RNA-associated DRAQ5 fluorescence in fixed
cells ŽSmith et al., in preparation.. Further applica-
tions derive from the cell permeant nature of the
agent with the advantage that it can be prepared as a
pure, stable, synthetic compound with defined spec-
tral properties. DRAQ5 also has the further advan-
tage of being soluble in biologically compatible sol-
vents. The agent belongs to a class of compounds
with defined cytotoxic mechanisms, the an-
thraquinones, permitting evaluation of the agent for
hazard monitoring purposes.
Terstappen et al. Ž1988. introduced the use of
LDS-751 to discriminate intact from damaged cells
in a flow cytometer, after fixation with paraformal-
dehyde. Three major cell populations with dim stain-
ing Žerythrocytes and platelets., intermediate staining
138 P.J. Smith et al.r Journal of Immunological Methods 229 (1999) 131–139
Žintact cells. and intensive staining Ždamaged cells.
could be distinguished. This approach was extended
to a method for immunofluorescence measurements
of cells by flow cytometry which does not require
washing procedures, permitting absolute enumeration
of cell subpopulations ŽTerstappen et al., 1989.. High
resolution analysis, approaching or surpassing that of
propidium iodide, does not appear to be feasible with
LDS-751 although it has been used for the quantifi-
cation by flow cytometry of function-associated anti-
gens on neutrophils and monocytes in unlysed, un-
fixed, peripheral blood samples ŽMcCarthy and
Macey, 1993.. These workers also pointed out the
advantage of avoiding potential artefacts that can
occur due to the use of fixatives, erythrocyte lysing
agents, or anticoagulants which are also divalent
metal ion chelators. These advantages also apply to
the use of DRAQ5 in whole blood preparations.
The comparison of LDS-751 and DRAQ5 reveals
the lack of DNA content discrimination achieved by
the former together with its significant ‘‘contamina-
tion’’ of emission wavelength regions below 600 nm
which would require electronic compensation. These
disadvantages were not apparent with DRAQ5.
We have recently determined that DRAQ5 inter-
action with DNA by intercalation is accompanied by
a red-shifted emission spectrum which potentially
enhances its utility as a far redrlow infra-red probe
if used in combination with visible range fluo-
rochromes ŽPJS; unpublished data.. Furthermore,
there appears to be little contribution to the fluores-
cence of whole cells originating from interaction
with RNA, and the resolution of DNA content distri-
bution is similar to that achieved by propidium io-
dide staining of fixed cells. Here we have demon-
strated its utility in the analysis of nucleated cells in
whole blood and density gradient-separated blood
cell preparations using a conventional bench-top flow
cytometer equipped with a standard low power argon
ion laser. DRAQ5 could also be considered as a
fluorochrome used in the calibration, standardisation,
and configuration of such systems given that no
compensation correction is required for DRAQ5r
FITC combinations. Furthermore, we suggest that
the introduction of bench-top systems incorporating
inexpensive red-light emitting sources will permit
the further exploitation of the novel fluorescence
signature of DRAQ5 in cellular studies. We foresee a
role for DRAQ5 in the expanding area of flow
cytoenzymology, particularly for the monitoring of
cell cycle-related enzyme activity or reporter gene
expression.
Supported in part by research grants from the
Association for International Cancer Research
ŽAICR. and the UK Medical Research Council.
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