Download SIMULTANEOUS STAINING OF RIBONUCLEIC AND DEOXYRIBONUCLEIC acridine orange

THE
JOURNAl,
Copyright
oc H ISTOCHEMISTRY
AND
© 1977 b’ The Histochemical
SIMULTANEOUS
C YTOCHEMISTRY
Society.
STAINING
ACIDS
IN
UNFIXED
Sloan-Kettering
Received
for
to chelating
agents
with
cells
is
procedures
May
10,
that
1976,
DNA
metachromatic
discussed
measured
in a flow
employing
acridine
in terms
of staining
provided
that
acridine
Center,
is denatured
RNA
to ensure
the
T. SHARPLESS
of deoxyribonucleic
is described.
Cells
staining
orange
AND
25. No.
1. pp 46-56.
1977
Printed in USA.
DEOXYRIBONUCLEIC
ACRIDINE
ORANGE
SYSTEM’
Cancer
publication
fixed, but permeable,
cells
ionic detergent
at low pH.
differentially
RIBONUCLEIC
Z. DARZYNKIEWICZ,
Memorial
cells
OF
CELLS
USING
CYTOFLUOROMETRIC
F. TRAGANOS,
Simultaneous
Vol.
Inc.
dye,
New
and
M.
D
York,
New
in revised
IN
R.
MELAMED
York
form
A FLOW
10021
July
14,
1976
(DNA)
and ribonucleic
acid (RNA)
in nonare made permeable
by treatment
with nonprior to, or during
staining,
by exposure
of
(native)
and RNA (denatured)
may be stained
acridine
orange.
The
fluorescence
of individual
cytofluorometer.
A comparison
between
various
staining
orange
or other
intercalating
dyes
in unfixed
cells
specificity,
cell permeability
and preservation.
Evidence
staining
of unfixed
cells
may
be used
as a simple,
fast
is
is
means
of
obtaining
information
on cell ploidy
levels
and cell cycle status
from DNA measurements
(green
fluorescence),
and cell transcriptional
activity
from RNA staining
(red fluorescence),
in human
and murine
cells lines, peripheral
blood and bone marrow
specimens
from patients
with leukemia
and mitogenically
(phytohemagglutinin)
or antigenically
(mixed
lymphocyte
culture)
stimulated
human
peripheral
blood cultures.
Exposure
of cells to detergent
at low
pH as an alternative
to cell fixation
or hypotonic
treatment
is proposed
as a fast, convenient
method
of making
cells permeable
to dyes.
Specific
staining
of’ nuclear
acid
(DNA)2
within
individual
deoxyribonucleic
cells
offers
means
level
of
their
analysing
distribution
cell
cycle.
in
Many
dyes that
Recently,
introduced
proteins
fixed
cells
using
DNA
with
stain
ing of DNA
correlate
the
position
We
and
the
various
of
cells
and
stages
methods
staining
are
of’ the
ent
employ
the
differentially
dye
propidium
fluorescein
for
stained
as
Simultaneous
ribonucleic
acid
(RNA)
by which
may
cycling
cells
activity
work
was
supported
by
U.S.
having
(G,,
based
on
ble
are
cells.
helical
G2
use
of single
nucleic
acids
It
compared.
cells
(G0) from
The
of’ acridine
stain
cycle
of differ-
transcriptional
M).
+
cell
dormant
increased
S,
the
differential
a
activities
be useful
to distinguish
a minimal
level
of transcription
he
to
its
method
is
(AO)
orange
stranded
as
versus
a
dou-
(2, 6, 7. 20).
MATERIALS
Public
AND
METHODS
Cells:
Human
peripheral
blood
separated
on ficoll-hypaque,
were
mal healthy
donors.
Lymphocytes
DNA
from
differen-
monocytes
Health
glutinin
Service
Grant
126CA14134
through
the National
Bladder
Cancer
Project.
2Abbreviations
used in text: DNA,
deoxyribonucleic acid;
P1, propidium
iodide;
RNA,
ribonucleic
acid;
AO,
acridine
orange,
PHA,
phytohemagglutinin;
MLC,
mixed
lymphocyte
culture;
ALL,
acute
lymphocytic
leukemia;
F550, green fluorescence
intensity
measured
in a band from 515-575
nm; F.,00,
red fluorescence
intensity
measured
in a band from
600-650
nm.
Stit.
(MLC)
after
6
days
(3).
(ALL)
tine
46
way
from
of patients
before
were
and
kindly
or
absence
as
previously
mixed
microtiter
No
attempt
with
acute
in
provided
plastic.
was
prior
made
to
Cells
cul-
remove
prepared
and
in
peripheral
leukemia
treatment
Doctor
to
sampled
lymphoblastic
by
(9).
cultures
were
marrow
VILO
and
described
plates.
bone
after
to
of phytohemag-
iradiation
preparations.
the
cells.
from norseparated
lymphcoyte
by
in
MLC
from
same
72 hr
for
cells
mononuclear
obtained
were
of adherence
inactivated
the
monocytes
blood
for
cells
svere
and
lack
presence
(PHA)
ilator
ture,
the
by
in the
cultured
This
unfixed
stain-
and
protein
makes
it possible
metabolic
state
of the cell with
in the cell cycle.
propose
a simple
method
individual
also
with
(P1)
isothiocyanate
proteins.
within
transcriptional
may
in
iodide
stained
The analysis
of RNA
in relation
to the
would
be of special
interest
when
cells
intercalate
into DNA
(4, 5. 13. 15, 23).
Steinkamp
and
Crissman
(4, 22)
a two-parameter
analysis
in which
and
counter
ploidy
of the
DNA
for
the
tially
a
with
Z.
Arlin.
vincris-
Cell
STAINING
IN
CELLS
USING
designated
CEM,
derived
from
a childhood
lymphoma
progressing
to ALL (12) and EL4, a murine
T cell leukemia
(19),
were
a gift of Doctor
P. Ralph.
Friend
virus-induced
murine
erythroleukemia
cells
ACRIDINE
47
ORANGE
were as follows:
two-step,
pH 3.0: Aliquots
(0.2 ml, containing approximately
2-5
x 10 cells)
were withdrawn
from cultures
and were added
to 0.5 ml of a solution
containing: 0.1% (v/v)
Triton X-100 (Sigma Chemical
Co., St. Louis,
Mo.), 0.2 M sucrose,
10’ M EDTA
and 2 x 102 M citrate-phosphate buffer, at pH 3.0
fluorescence
is measured
in two
F530 (515-575
nm) and F>,00 (600650 nm) as the cells travel in single file fashion through
the focused
beam
of an argon
ion laser (488 nm),
subtracted
from background
fluorescence,
digitized
and recorded
for further
analysis.
Staining
specificity
with AO: Lymphocytes
from
PHA cultures
(48 hr) were treated
for 1 mm with a
solution
consisting
of 0.1% (v/v) Triton
X, 0.2 M
sucrose and 20 mM citrate-phosphate
buffer at pH 3.0.
The cells were then centrifuged,
suspended
in 0.25 M
sucrose-5
mM
MgCl2-20
mM
tris
HC1 (pH 6.5) and
incubated
for 20 mm at 37#{176}Cin the absence
of nucleases
or with 10 units/ml
of RNAse
A (Worthing-
(9).
ton
lines
(10)
were
Doctor
subcultured
from
a cell
line
maintained
by
C. Friend.
Cell
treatment
and
staining:
The
various
staining
protocols
1. AO:
Triton
dures
X-100
at the
was
included
indicated
pH
in the
to increase
various
cell
proce-
permeability
yet
maintain
cellular
integrity
(see
Discussion).
The chelating
agent
EDTA
was used to facilitate
RNA denaturation
as previously
described
(7, 9). The
cellswere stained one minute later by addition of 1 ml
of a solution
containing
0.002%
(20 g/ml)
AO (Polysciences
Inc., Warrington,
Pa.), 0.1 M NaCl and 102
M citrate-phosphate buffer, pH 3.8 (9, 11). Cations
were
included
in the staining
mixture
to ensure
staining
specificity
(see
Discussion).
The
final
AO
concentration
was approximately
4 x 10 -5M.
IL AO: two-step,
0.1 N HC1: Aliquots
of 0.2 ml of
cells in media
with 10% fetal calf serum
were
mixed
with 0.3 ml of a solution
containing:
0.1% (v/v)
Triton
X-100,
0.1 N HC1 and 0.15 N NaC1. After mixing
and equilibration
(about
15 sec),
1 ml of AO (5
g/ml)
in 5 x 10
M EDTA,
0.15 N NaCl, 0.1 M
phosphate-citrate
buffer,
pH 6.0 was added
giving
a final
dye concentration
of 1.1 x 10
M AO.
III.
AO:
one-step:
containing:
10cells
5 tg/ml
M EDTA
2
in
serum
P1:
for 5 mm
resuspended
in
0.05
after
P1:
two-step
except
that
dye
previously
(Data
multaneous
cence
individual
low
were
cells
Southboro,
of green
(1-19#{176}) forward
in
suspension.
as
diploid
mean
The
sys-
to that
flow cytoSystems
Inc.,
Mass.)
minicom-
provides
and
red
light
As
population
in cell
decreased
AO
with
red fluorescence
By
use
specificity
a
in
lished
(9).
the
and
by
increased
the
red
C
was
cells
could
to
up
(9).
16
hr
a marked
could
be identified
green
(DNA)
fluorescence
in
all
the
G-S
of
boundary
in
0.2
C2
green
tg/ml
were
the
of
stimulated
increase
of
terminated
M populatin
-+-
as a population
level
system
hydroxyurea
days
cultures
increase
which
as analysed
2 mM
addition
before
hr
synthe-
by treatment
no
The
24
RNA
in this
at
fluorescence,
gluorescence)
vinblastine
pile
estab-
added
delineated
stimulated
cultures
with
24 hr of cultures.
By three
red
ig/ml)
transition,
PHA
after
seen
staining
was
fluorescence,
population
be
agents,
prevented
G0 to C
(RNA)
of
of the
(9).
(0.05
of cultures
as dyes
87%
cultures
D
the
is a result
while
blocking
stimulated
Actinomycin
therefore
The
of
PHA
initiation
sis
DNA
to RNA
variety
and
detergent
as well
fluorescence
cellular
is due
of
88%
with
to nucleases
88% of the green
interaction
by
treatment
permeability
and at least
after
Freehold,
I (Worthington).
F>,00 by 7%. Therefore,
results
at twice
of the
C0
(or
C)
the
cells
(9).
RESUI.TS
One-step
and
1220
Corp.,
DNAse
was
model
to a Nova
the
N NaCI,
in principle
(Bio/Physics
N.J.)
or with 4 x
After centrifugation
the
cells
were
resuspended
in an AO solution
(staining
method
I) and the mean F,30 and F>,,0 for the
diploid
cell population
was calculated.
Cells treated
with
RNAse
showed
a minimal
(3%) decrease
in F,30
but an 87% decrease
in F,,00 when compared
to control
cultures.
In the presence
of DNAse,
the mean
F,30 of
units/ml
N HC1
mg/ml
of fluorescence:
interfaced
angle
treated
in 0.1
0.15
Biochemical
10
caused
buffer.
pH 6.0.
(6). A research
General,
contain-
citrate
Cells
is identical
measurements
and
solution
cenwere
the
bands,
(increased
were
pellets
of 0.05
buffer,
FC2000
N.Y.),
M).
in 5 x 10- 3M EDTA,
study
X-100,
( 18),
wavelength
(9).
added
to 0.2 ml of
10%
fetal
calf
procedure
a concentration
described
fluorometer
Mahopac.
puter
Triton
sodium
N HC1:
measurement
in this
solution
(13).
staining
M phosphate-citrate
Analysis,
the
0.1
AO
for AO
used
AO
(v/v)
iodide
in a 0.1%
of Krishan
P1 at
substituted
tem
an
1.2 x 10
suspensions
150 g and
the cell
at
two-step,
in the
0.1
0.07%
a propidium
mg/ml
the method
V.
of
AO concentration
one-step:
Cell
(final
trifuged
AO,
ml
and 0.15 N NaC1 was
with
approximately
media
IV.
ing
0.6
described
si-
scatter
previously
B shows
staining
wide
for
AO
a comparison
procedure
use
using
employing
and
a technique
a DNA
stain
of
from
a patient
with
ALL
blood
suspended
phate
buffered
saline
and
10%
AO
solution
detergent
cells
human
mixed
peripheral
with
an
(staining
permeable
method
to
the
dye
III)
in
(13).
A
leukocytes
in
fetal
and
1A
a single-step
AO and
P1 as
Figure
P1:
between
sample
was
fluores-
staining:
calf
phosserum
containing
to
to
make
a chelating
the
48
TRAGANOS
ET AL.
cells
while
with
little
higher
. red
cycling
CV 7 3
B
compared
IL
C
0
(I,
a,
U
be
CV 8.3
using
due
lack
to
lysis
1. Computer
and
RNA
drawn
distribution
shift
cells
(AU.)
histogram
of peripheral
of the
blood
DNA
not
leukocytes
and
Methods).
B, DNA
distribution
of cells
stained
with P1 in 0.1% citrate
(13) (staining
method
DNA distribution
of cells treated
with 0.1N
HC1 prior to staining
with 0.05 mg/ml
P1 in 5 x 10’
M EDTA,
0.15 N NaC1,
0.1 M phosphate-citrate
buffer,
pH 6.0 (staining
method
V). The coefficient
of
variation
(CV)
for the G, population are indicated.
IV).
C,
agent
(EDTA)
which
preferentially
hypotonic
microscopic
(Table
IV)
after
(Fig.
of’ AO
6 hr
(Fig.
This
A
stained
in the
shift
stained
conditions
(Fig.
examination
more
2).
of culture
2A).
preparation
AO:
staining:
and
red
of three
(staining
method
conditions
which
be
was
with
P1
2B). Fluoresof specimens
I).
Two-step
hypotonic
(staining
cells
could
both
ways
revealed
that
the detergent
cells
remained
intact,
while
cells
in a hypotonic
medium
were extremely
the vast
majority
of chromosomes
apfree
or in small
clumps
on the slide
(Figure
3A-C)
from
a patient
gent
solution
solution
and
pro-
IV) to deterabove
might
of mitotic
distribution
in the
any
double-stranded
RNA
(7, 9) (Fig.
1A).
Staining
was
performed
at pH
4.0.
Parallel
samples
were stained
with P1 in the presence
of
citrate
the
staining
III and
noted
hypotonic
observed
observed
treated
treated
stained
fragile,
pearing
one-step
loss
of colcemid
scatterplots
histograms
denatures
two
methods
differences
and
in
was
under
cence
from a patient
with ALL. A, green (DNA,--)
and
red (RNA,
) fluorescence
distributions
of cells
stained
by the one-step
AO procedure
at pH 4.0 (see
Materials
suggest
M).
+
membrane
to
presence
FIG.
the
a nuclear
major
intensity
(G0)
values
S + C2
+
cedures
(staining
mine
whether
the
susceptible
Fluorescence
activity
fluorescence
Logarithmically
growing
Friend
virusinduced
murine erythroleukemia
cells, cultured
in the absence
and presence
of colcemide,
were
0
0)
C
(G1
cells
transcriptional
method
(RNA)
tissue
Figure
3
shows
and
green
(DNA)
culture
cell
lines
and
a BM sample
(Figure
3D
with ALL treated
with
a deterin 0.1 N HC1 prior
to staining
II).
(Fig. 1B). The distribution
of cells within
the cell
cycle
(Fig.
1A and B, solid lines)
are reasonably
similar
for the two methods,
though
fewer cells
(13.9
versus
16.4%)
thetic
(S),
stages
of
premitotic
the
cell
treated
with the hypotonic-citrate
ure
shows
1C
appear
cycle
the
same
detergent
in 0.1 N
with
P1 as in Figure
The
distribution
the
cell
Figure
cycle
1A,
cycle
status,
and,
the
therefore,
ity of a cell.
distribution
followed
(staining
within
information
AO
staining
red
which
stages
17.0%
0)
C
F530
(A
U)
(A
U)
0
‘C
U
0)
(-)
of
observed
in
of
the
aoo
M compart-
+
regarding
procedure
fluorescence,
relates
to RNA
to the
0
with
various
G2
5)
by staining
method
V).
to that
in the S +
syn-
pretreated
approximately
to the
second
parameter,
line,
Figure
1A)
(7)
HCI
lB
DNA
mitotic
(M)
preparation
solution. Fig-
cells
of cells
with
the
and
in the
is comparable
cells distributed
ments.
In addition
in
(G2)
transcriptional
The
major
peak
to the
(Figure
1A)
probably
cell
offers
(dotted
staining
activ-
left of the
represents
a
FIG.
2. DNA
distribution
of FL cells
before
and
after
a 6 hr colcemid
block.
A, green
fluorescence
(DNA)
histogram
of logarithmically
growing
(--)
and
colcemid
blocked
(----)
cells
stained
with
AO by
the one-step
procedure
at pH 4.0 (method
III). B, red
fluorescence
(DNA)
histograms
of cells
(as
in A)
stained
by the
hypotonic-PI
staining
procedure
(13)
(staining
method
IV). Note
the apparent
lack
of increase
of cells in the C2 + M phase
when
treated
with
the hypotonic
citrate
solution.
STAINING
IN
CELLS
USING
ACRIDINE
TABLE
Morphologic
and
Staining
I
Characteristics
Hypotonic
of Unfixed
or Detergent
Cells
Microscopic
Interphase
evaluation
by
Permeable
Detergent
l’reatment’
-
-
citrate)
Lyse;
cells
Made
Treatment
Hypotonic
Treatment
o.U;
49
ORANGE
pH
Lyse;
nuclei
separate
in
pH
7.()
nuclei
4.0
pH
Unbroken
Unbroken
Intact;
break
under
MS.#{176}
Stable
3.()
pH
I.))
Jnbroken
agglutinate
suspension
Metaphases
Stainability
with
AO
Broken;
chromosomes
free or in
clumps
Broken;
chromosomes
free or in
clumps
Stable
pH3.0c
pH6.0c
PHl.0j6.0(
Nucleoplasm
green
green
green
yellow
green
yellow
green
Nucleoli
poorly
delineated
red, when
present
Metaphase
cells
present
in
some, absent
red
red
red
red
red
red
remnants,
red
Metaphase
absent
red
red
red
red
red
Cytoplasm
Cytofluorometry
(AO or P1)
Metaphase
cells
present
cells
Metaphase
present
cells
Metaphase
cells
present;
2.4
x higher
F,30
in others
Cells
were
made
Materials
and
o Sensitivity
sec) or after
Cells
sity,
permeable
to
Methods.
to mechanical
application
treated
the
stress
stain
by
(MS.)
was
detergent
evaluated
with
(Fig.
3A)
logarithmically
cells
while
S phase
line
derived
(12)
appears
levels;
the
on normal
(not
shown),
G2
from
+
a
to
and
the
line
growing
proportion
M
(Fig.
patient
contain
3C).
The
blood
upper
of tetraploid
cells
distribution
of the two
have
a
and “tetraploid”
from a patient
uniform
the
be
at
twice
a
tively
few
red (RNA)
the
G1
cycling
cells
fluorescence
population
fluorescence
minor
population
scriptional
activity
is a
of’
cells.
A bone
marwith ALL appears
to
DNA
distribution
(Fig.
3D).
histograms
only)
However,
(in this
is skewed
intensitities
of cells
(dotted
with
with
line,
mitogenic
(DNA)
pH
3.0
in studantigenic
peripheral
lymphocytes
3 days
in culture
is still
(RNA)
fluorescence
and
(G0),
(DNA)
but
green
C2 + M)
in mitosis
(Fig. 4C).
was ob-
served
cultures
in which
this
in parallel
indicating
staining
described
olds
19 hr prior
it
percentage
dead
cells
that
procedure
(9),
by setting
is a simple
(G1
an
in S and
of cells
added
fluorescence
increased
of
moder-
intensity
green
in
a locus
fluorescence
(cells
An accumulation
4D),
tran-
at
used
and
there
low red
(RNA)
both
(10
human
After
unchanged
red
with
vortexing
4A) occupy
a locus
of
moderate
green
(DNA)
PHA,
with
in
cells),
and
red
the
case
a
of
with
increased
and
cells
and
intensity.
ate
green
(Fig
described
indicated.
Unstimulated
cultures
(RNA)
presence
as
normal
lymphocytes.
cells
vigorous
(PHA)
of
cells
was
toward
after
as
with
detergent
to staining
was
response
noncycling
rela-
suggesting
increased
Fig. 3D).
the
fluorescence
to
of fluorescence
which
overlapping
distributions
of
in control
low
red
lymphocytes
appears
samples
X-100)
under the microscope.
at pH 1.0, 3.0 or 6.0
(MLC)
blood
with
lymtwo
ploidy
Triton
Cell
treatment
immediately
prior
ies
of
superimposable
peripheral
population
“diploid”
row sample
higher
cell
green
fluorescence
intensity
(Fig.
3B). The
fluorescence
histogram
in Figure
3B shows
a
broad
result
for
EL4
a greater
is reasonably
human
cycling
the
red
the
and
CEM
phoma
lower
by observing
to a drop of cell suspension
at pH 3.0 or 1.0 and then stained
detergent
contains
in
(0.5-0.1%
of a coverslip
With
FL cultures
grown
to a high
cell denthe majority
of cells are in the G, phase
of
growth
treatment
to analysis
mitoses
are
(Table
the
matter
vincristine
at 3 days
(Fig.
preserved
in
I). As previously
appropriate
to
of cells
in G0, C,, 5,
from such
a distribution
thresh-
quantitate
G2
+
(Fig.
the
M and
4E).
#{149}0
0
j05
_
FIG.
3.
sample
with
Computer
from
AO, at a final
two-dimensional
origin
represents
cells
A,
drawn
a patient
fluorescence
text).
ALL.
concentration
distributions
the relative
as measured
Friend
scatterplots
with
leukemia
(DNA)
C, logarithmically
and
All
cells
of 1.1
grown
distribution
growing
to
Each
a high
that
appear
EL4
cells.
cell density
as in A. D, a bone marrow
(DNA)
levels but the red fluorescence
suggesting
a small
population
and
of dots whose
horizontal
red (RNA)
and
green
by flow cytofluorometry.
cells
DNA
of C,
cell
with
scatterplot
with
higher
of three
cell lines
solution
The scatter
in 0.1
plots
is an accumulation
B,
to represent
Compare
histograms
a detergent
method
II).
and
a bone
N HCI prior
are computer
(abscissa)
and vertical
(ordinate)
displacement
(DNA)
fluorescence
intensity,
respectively,
density.
sample
from
distribution
cells
RNA
were treated
10’
M(staining
CEM
cells with
the
cell
cycle
cells.
Note
of approximately
the
two
sharp
diploid
and tetraploid
distribution
with
that
peaks
amounts
of cells
marrow
to staining
generated
from the
of individual
3 x 10 cells.
in the green
of DNA
grown
a patient
with ALL. Few cells are seen at elevated
of the C, cells appears
skewed
to increased
RNA
RNA
50
content.
(see
to a high
green
levels
STAINING
Mixed
using
for
lymphocyte
the
same
were
procedure
4B).
The
fluorescence
to that
may
be
(Figure
USING
analysed
after
obtained
is equivalent
PHA
cultures
and
6 days
bution
with
cultures
staining
IN CELLS
intensity:
conditions
increases
the
distriobserved
similarly
cations.
Thus,
ence of 5 mM
for DNA
the
green
binding
dyes
by
blood
ALL
are
(staining
in
0.1
method
II).
(10).
intensity
lymphocytes
from
in
There
at
pH
4.0
(staining
at
low
Specificity
of
AO: Acridine
intercalating
staining
propidium
tion
with
iodide
nucleic
staining
and
teins,
several
the
dye
possible
per
site
sites
AO
per
specificity
staining
dye
of
precludes
is
higher
than
for
former
are
the
AO
pref-
concentrations
the
must
(1,
amount
be
staining.
the
DNA
quan-
their
F, Corski
Rigler
(20),
to increase
Acetylation
proteins
A, Darzynkiewicz
In preparation.
of
prior
interaction
several
rehydration)
MR:
of
precisely
to
with
steps
and
is time
to
stacking
dye
cells
RNA
(7)
all
double
involved
RNA
all
DNA
in
heating
have
also
at
agents
effect
of
destroyed
cations,
ribosomes
AO
staining
that
either
EDTA
(or
the presence
AL)
in
as
(24).
of
filled
may
cell
treatment
to stainwhile
the
strength
is lower
in
to
change
be
in
achieved
temperatures
in
by
(7).
with
room
We
chelat-
temperature
it
of
is
Thus,
DNA
when
in the
situ
a result
just
selectively
significant
at
of
all RNA
in situ,
while
it does
(7). Apparently,
the secondary
rRNA
as
50%
stranded
it is possible
DNA,
This
EDTA)
DNA
of
pairing
moderate
almost
structure
Since
in base
meta-
least
to
helical.
without
(i.e.,
denatures
due
red
double
necessary
than
RNA
found
at
the
at 530
nm)
stranded
RNA prior
is single
stranded,
conformation.
cell
Since
in
RNA
bonds
denature
manifested
is
single
helical
remains
of
(488
interactions
it
that
case
helical
acid-dye
dye-dye
is
ing
DNA
double
on
the
RNA.
of nucleic
stacks
(1).
double
of the
to use
the
situ
RNA:
prop-
versus
AO
nm)
in
any
such
versus
unique
an
DNA
type
are
denature
tor
when
in green
fluorescence
excitation
with
blue
and
conformation
Z, Sharpless
be
digestions
with
the specificity
into
this
(640
cellular
the
con-
be-
should
in order
stain
contrast,
acids
cell
(dehydra-
It
has
intercalates
upon
in
ing
greatly
interaction
of DNA
be employed
and
nucleic
the
be
detectable
critical
AO
RNA:
results
(16)
not
involves
tion,
acetylation,
suming.
3Traganos
of
sample.
of
procedure
T, Melamed
affinity
approach,
proposed
by
of the acetylation
reaction
amino-residues
This
are
the
sites,
phosphate
the
in each
A second
makes
use
There
this.
this approach
is hard
to impleto maintain
a constant
molar
ratio
for
titated
pro-
Since
AO
at low
DNA
available
the
for
stained
16). However,
ment,
since
doing
(26).
binding
erentially
be precluded.
of
may
chromasia
mostly
of
control
to assure
differentially
AO
light;
electrostatic
to
the
acids.
staining
interaction
with
spe-
result
is especially
of
acid
nm
involves
the use of AO at low
that
is at a low molar
ratio
of
intercalating
of
must
binding
electrostatic
the
to
Namely,
in its interacto its positive
polyanions,
ways
One approach
concentrations,
AO
acids,
between
that
dye
nucleic
interacts
electrostatically
(1). Therefore,
to obtain
of nucleic
interactions
erty
with
macromolecules
all
nucleic
Differential
than
other
bromide,
acids
specific
ethidium
or mithramycin
acids.
Due
AO also
polyanions
cific
of nucleic
orange
is less
dyes,
such
as
the
however,
that
are required
Denaturation
DISCUSSION
the
appears
virtually
and
This
(Nat
or Mg2)
AO (10’
M) for
to contain large quantities of polyanions
glycosaminoglycans)
are stained.
(i.e.,
pH
5).
charge,
other
is
AO
of
acids
and
fluorescence
known
in green
treatment
(14)
of staining.
deter-
cations
with
staining
nucleic
stressed,
nucleases
between
AO and
if cells are stained
in the presence
of
cells
may be stained
in the presMgC12
or 0.1-0.15
N NaCl
(6, 9).
sites
than
tween
a patient
increase
after
to
when
with
anionic
impeded
due
DNA
one-step
is a 2.4-fold
intensity
5 shows
(F,30)
in
51
Under
conditions
where
are in excess,
competing
other
sites
Figure
III) or first treated
HC1 prior
to staining
N
fluorescence
binding
intercalation
stained
method
(Fig.
available
fluorescence
peripheral
gent
of
intercalating
AO
with
Treatment
of cells at low
known
to extract
histones,
number
ORANGE
A highly
specific
interaction
nucleic
acids
also is obtained
in equilibrium
with
the dye
culture
analysed3.
Staining
pH under
ACRIDINE
in
the
divalent
of
isolated
for
RNA
cells
cell
of
case
conditions
versus
intact
chelation
in
are
is
differential
cells
are
pretreated
fulwith
citrate),
or when
they
are stained
of these
chelating
agents.
concentration:
The
differential
staining
other
significant
of
double
in
fac-
versus
MLC-6
Days
,-‘
r
-
i.
-
r:-
...
1
-
I
:
.4
#{149}1
/
1TT
---
-
-
I
0
100
50
PHA
-
50
3 Days
--,
;fr.
.-,
.:*
t’
..-..
I
I
‘:
.
‘
,
-
-
-
-
-
-
-
SI
tUl
00
FIG.
4. DNA
and
RNA
distribution
in control
detergent at pH 3.0 and then stained with AO
and
red
(RNA,----)
fluorescence
histograms
and
stimulated
(staining Method
of human
peripheral
52
human
leukocytes.
The
I).A, scatterplot (as in
blood
leukocytes
cells
Fig.
cultured
were
3) green
for
treated
with
(DNA,--)
48 hours
in the
STAINING
IN
CELLS
USING
5)
0
x
(32
0)1
C
I)
(77
0
‘C
Fluorescence
(A U)
Difference
in green
(DNA)
fluorescence
of cells treated
at pH 4.0 or in 0.1 N HC1 and
with
AO. Peripheral
blood
leukocytes
from
a
with
ALL
were
stained
by the one-step
(III)
intensity
stained
patient
AO
method
0.1
N
or first
(-)
HC1
(II)
followed
treated
by
with
staining
detergent
with
optimal
--).
all
nucleic
As
we
the
same.
acids
have
is the
shown
concentration
sites
dye
Ref.
that
are
F,30
approaches
staining
is
done
in
at
a
the
at
presence
of the
N NaCl
cations.
With
the optimal
between
5 x 10’
varies
Considering
the
concentration
binding
which
of
absence
of
nucleic
concentration
DNA
phosphate
Cell
makes
per
sample
an
cation,
of
low
molar
the
single
as
and
excess
the
of fixed
cluding
centrifugation,
cells
involve
some
cell
cells
on the
other
rameter
cells
to the
can
(see
for
help
18)
of
nucleases
Hypotonic
introduced
treatment:
a technique
propidium
to break
nuclei.
according
not have
integrate
dye
fluorescence
analysing
molar
or
treated
ment.
vides
AO/
DNA
(13)
tion
on the
since
some
or all
Loss
of
cytoplasmic
and
cell
types
Krishan
(13)
of nonfixed
a hypotonic
thus
makmethod
staining
microscopy,
Table
colcemid
or
used
to obtain
in G2
escape
ymca
cells
treat-
method
proof cellular
of cells
may
I).
cultures”
of metaphase
the hypotonic
M cells
disor
within
the remobserved
under
“synchronized
be
is
of
such treatthese
cells do
while
the hypotonic
and
uniform
staining
proportion
the
they
totally
chromosomes
(as
with
it cannot
assess
the
hypotonic
treatauthor,
“results
in
a subpopulation
detectable
using
AO
stain-
some
dye.
The
uniform
clumped
membrane
when
Thus,
rapid
dead
iodide
using
membranes,
cell
or UV
double
pa-
from
to
a nuclear
envelope,
leaving
either
isolated
cultures
This
living
(13). Unfortunately,
mitotic
cells;
since
chromosomes
of the cell
informa-
uptake.
controls
However,
to the
some
nants
alkaloids,
was not
of nonfixed
provides
Recently,
for staining
phase
an
Staining
classify
as
of staining.
diploid
ment,
inand
of dye transport
into the cell
in lysosomes
(17). It does
staining
of nucleic
acids
or
specificity
cells
with
treatment
steps,
consuming
hand,
or
the
Unfortunately,
differentiate
Ref.
nude-
evaluate
several
loss.
in vivo
Thus,
for
agents
optimal
use
(7).
is time
living
to chelating
to
requires
may
These
exogenous
possible
on
(7).
only
to
staining
AO
However,
at
it
staining
related
not
also
of AO
optimum
to be
to chelating
or
of
M.
3 x 10-’
M and
ratio
near 0.4 (26).
bases
on
10’
versus
appears
The
fixation:
4 x
(8, 26).
acids
as
and
phosphate
permeability
Cell
of
isolated
nuclei”
ment
also lyses
concentraand
conditions
any
the
Since
competing
(AO/DNA
staining
stranded
but
established
acetone
ing cells
permeable
to the
rapid
and gives an excellent,
5 mM
MgCl2
concentration
is always
those
differential
AO:
number
site
under
the
cell
there
(6).
plateau.
cations
(Mg2
or Nat),
this optimal
tion of AO depends
on the concentration
the affinity
0.10-0.15
which
saturated
range
in
the
by a plot of F,30 versus
26), and is indicated
concentration
change
concentra-
previously,
is
intercalation
This
may be determined
AO concentration
(see
per
which
tion
were
permeable,
the
the
DNA
alcohol:
AO,
approximately
dye
nearly
ratio)
fully
and
fluorescence
intensity
for the C0/C,
by 2.4 times
(32.1
to 77.8)
after
at low pH, the coefficient
of variation
(CV)
of’ AO.
in
(--
vs.
in
based
on the rate
and its accumulation
not permit
selective
stranded
tion
AO
in
AO
the mean
increased
treatment
remained
by
intensity
5.
single
are
RNA
specificity
ci)
C.-)
While
cells
prefixed
ases,
(I)
FIG.
of
cells
cells
53
ORANGE
ability
agents
cv491\
0V48
C
8)
ACRIDINE
informaM phase,
+
detection.
components,
including
absence
of stimulant.
Note
moderate
green
(DNA)
and low red (RNA)
fluorescence.
B, mixed
lymphocyte
culture stained
as in A. Cells
with
increased
red (RNA)
fluorescence
(C,)
or increased
green
(DNA)
and red (RNA)
fluorescence
(S + C2 + M) can be observed
in addition
to C0 cells.
Cells
with
decreased
green
fluorescence
represent
dead
or dying
cells.
An additional
population
to the left of the C0 cells has been
thresholded
out for clarity and will be discussed
in a future publication3.
C, human
peripheral
blood leukocytes
cultured
for 3 days in
the presence
(B). D, cells
accumulation
cence
histogram.
fluorescence
chronous
of PHA
(9). The distribution
of cells is very similar
to that observed
in mixed lymphocyte
cultures
as in C in which 0.2 og/ml
vincristine
was added
19 hr prior to analysis
at 3 days. Note the
in the C2 + M phase of the cell cycle in both the scattergram
and the green (DNA) fluoresD, lymphocytes
cultured
with PHA for 3 days. The thresholds
for red (RNA) and green (DNA)
cultured
of cells
are
cultures
masked
were
to discriminate
used
to identify
cell
the
subpopulations.
positions
Control
experiments
of the subpopulations
and
with
thresholds,
blocking
agents
as presented
or syn-
(9).
54
TRAGANOS
RNA,
ment
ET
the
also precludes
using
the hypotonic
treatfor RNA
staining
of interphase
cells.
Treatment
sure
with
of
cells
known
brane
to
detergent
at
detergents
at
to induce
disintegration
and
subsequent
methods
of isolation
phenomenon.
lyse when
also
noted
are
with
detergent
the
nucleoli
clei from
lysed
cells stain
I), suggesting
that
RNA
red
does
levels
of
F>,00
differ
from
membranes,
which
stains
red,
but
are
made
the cytoplasm
while
(Table
There
the
permeable
procedure,
by
the
with
have
do not
ways
detergent.
dye
(AO
P1)
denaturing
agent
(EDTA)
are
single
solution;
the pH of that
tained
at 4.0.
This
since
we
promise,
at
the
pH
below
interaction
acids
at
pH
the
above
a comwith
AO
the
specificity
of
dye
and
nucleic
cells
lyse.
Unfortunately,
although
cells do not lyse
4.0, they do appear
to be fragile
and break
4.0,
at pH
down
after
additional
mechanical
pipetting
or mixing.
Thus,
the
one-step
seems
to
coefficient
the
for
possible
ing
during
the
flow
cell
mechanical
is
like
the
the
simplest
resolution
(the
it requires
lysis
under
vigorous
fact that
and
lowest
careful
forces
con-
of shear-
raised.
retains
stress,
procedure,
at pH
are
and
at higher
pH.
During
the
concentration
of detergent
dye solution
is added,
and
Under
such
-treatment
its integrity
and resistence
yet
it becomes
nucleic
permeable
It
is
the
to
to
cells
from
absence
(i.e.,
in saline),
of
for
a several
Triton
acids
is carried
therefore
which
as a result
(e.g.,
X
(0.02-
out
at pH
unlikely
may
have
of RNA
that
been
3.0
any
solubilized
denaturation)
are
lost
due to their
insolubility
under
acid conditions.
When
detergent
treatment
is carried
out
0-4#{176}Cat pH
nous
3.0
or below,
nucleases,
appear
which
to be
any
may
effect
have
precluded.
We
at
of endoge-
been
released,
have
observed
that
in experiments
in which
the time
of detergent
treatment
was
varied
from
1-5
mm
no
alteration
in the mean
fluorescence
intensities
could
shown)
be observed
suggesting
cationic
Most
for the cell
that nucleic
and/or
DNA
is
acid-soluble
ble, stainable
types
stage
types
studied
(not
acids
are not lost
or nucleolytic
versus
total
intercalating
unavailable,
proteins.
DNA
dyes
being
The
portion
and
in some
cell
of genome
activity
above,
stances
extent
varies
types
(26).
it would
appear
to be advantageous
DNA
rather
than
in
attack.
DNA:
isolated
with
(10, 20,
masked
the
26).
by
of the availabetween
cell
is related
Considering
to the
the
under
some
circumto stain
all nuclear
only
the
unmasked
portion,
thereby
providing
a more
constant
(although
still relative)
measure
of DNA
per cell. By this
approach,
the differences
in chromatin
composition
or conformation
may be eliminated,
which
may
ties
the
cells
3.0 or lower
in their
treatment
below.
be
DNA
in a cytofluorometer.
two-step
with
detergent
then
are
stained
staining
step
the
drops
as the buffered
the
pH
membrane
the
stress
despite
is
best
of variation)
trol
In
treated
procedure
offer
or
all
directly
serum.
Serum
promembranes
against
ofcells
concentration
for
X-100,
make
taken
Only
a portion
of DNA
in situ
or
chromatin
is available
for interaction
RNA
into a
is main-
staining
to
are
either
through
“leakage”
Staining
of unmasked
cells
a one-step
and
represents
that
3.5 decreases
between
the
while
green
combined
solution
procedure
found
to AO
deeply
disintegrate.
to stain
In
or
in
detergent
ruptured
fluoresces
sufficient
they
chosen
(Triton
is adequate.
also
permeable
and nucleoli
nucleoplasm
I). Cells
in mitosis
are two
possible
made
seen
lower
Detergent
nu(Table
out of
those
nucleases.
detergent
minimal
Therefore,
0.04%)
them.
In these
preparations
of broken
cells (plus
isolated
nuclei)
mitotic
cells
are lost,
and the
intensity
of F>,00 and proportions
of cells at various
yet
when
suspensions
fold
above
AO
leak
was
v/v),
staining
to be lysed.
with
not
parallel,
unbroken
cell preparations.
We observed
that
cells treated
at pH
4.0 and
below
do not
0.07-0.1%
detergents.
of isolated
of the
cultures
containing
10-15%
teins
markedly
stabilize
cell
on this
at pH
as to exogenous
experiments
permeable
by phasethat
cells
all cells appeared
that
is
memVarious
experiments,
and
I),
as well
concentration
these
pH
cell
based
controlS
fluorescence
(Table
treated
4.0; at neutral pH
We
cell
in
UV
neutral
dye
The
Expo-
pH:
of the
lysis.
of nuclei
Indeed,
we observed
by
interference-microscopy
low
AL.
As
cells
gent,
particularly
content
helpful
of cells
or of different
described
with 0.1
removes
proteins
and
intercalating
types
above,
N HC1,
histones
in the
and
increases
the
sites
reacting
fold.
Despite
this
rather
remain
in
relatively
and
do
not
assaying
genome
the
activi-
in the same
sample.
treatment
of nonfixed
cells
condition
when
of different
presence
of deterother
acid
soluble
maximum
with
harsh
good
aggregate.
dyes
number
by
treatment
of
2.4the
morphologic
We
want
to
STAINING
stress,
the
that
however,
DNA
the
acid
to prevent
treatment
should
cold (0-4#{176}C) (25).
Treatment
of cells
significantly
with
change
AO.
Therefore,
regarding
sired
to
(i.e.,
tion,
with
genome
detergent
step
procedure
of the
correlate
at
pH
staining
cells
with
of analysing
on
a
normally
required
is avoided,
in
ploited
tion
this
on
the
(21,
for
cell
hand
With
The
AO staining
plied
to cell cycle
perturbed
mia,
of
and
Since
fixation
produced
and
on
mea-
additional
parameters
several
apcell
cycle
with
leuke-
blood
leuko-
cell-cell
interaction
be pointed
procedures
have
lymphoid
cells.
those
containing
of fibroblastic
out,
been
however,
tested
Other
Exposure
dure
of cells
for
making
digestion
to detergent
is a more
cells
treatment.
method
be
Kurland J, Traganos
MAS: In preparation.
at
low
permeable
or
care-
pH,
as
procethan
is the
Therefore,
we
used
in conjunction
suggest
with
F, Darzynkiewicz
the
CITED
MK:
JD,
Aggregation
Proc
Natl
Melamed
F, Sharpless
MR,
Acad
of
dyes
Sci
USA
Darzynkiewicz
T, Good
lymphocytes
and
staining
Z,
RA: Quantitation
by flow
phytohaemagglutinin
methods
acid and protein
chem Cytochem
Crissman
minutes.
cytofluorime-
by
flow
cyto-
for analysis
of deoxyribonucleic
in mammalian
24:64, 1976
HA, Tobey
RA:
Science
184:1297,
cells.
Cell cycle
1974
J Histo-
analysis
in 20
6. Darzynkiewicz
Z. Traganos
F, Sharpless
T,
Melamed
MR: Thermal
denaturation
of DNA in
situ
as studied
by acridine
orange
staining
and
automated
cytofluorometry.
Exp Cell Res 90:411,
1975
7. Darzynkiewicz
Z, Traganos
F, Sharpless
T,
Melamed
MR: Conformation
of RNA in situ as
studied
by acridine
orange
staining
and automated cytofluorometry.
Exp Cell Res 95:143, 1975
8. Darzynkiewicz
Z,
Traganos
F,
Sharpless
T,
Melamed
MR: DNA denaturation
in situ. Effect
of divalent
cations
and alcohols.
J Cell Biol 68:1,
multiparameter
73:2881,
to deter-
advantageous
Nager
of
fluorometry.
II. Comparison
with “C-thymidine
incorporation.
Clin Immunol
Immunopathol,
5:
326, 1976
4. Crissman
HA, Oka MS. Steinkamp
JA: Rapid
on
especially
large
amounts
of polyanions
or epithelial
origin,
require
above,
hypotonic
that
this
types,
Robin
preparation
1976
these
predominantly
cell
ful controls
utilizing
nuclease
mine
staining
specificity.
discussed
that
Miss
the
Wolf
9. Darzynkiewicz
Melamed
MR:
systems.
It should
or hypo-
try. Clin Immunol
Immunopathol
4:209, 1975
3. Braunstein
JD, Melamed
MR, Sharpless
TK,
Hansen
JA, Dupont
B, Good RA: Quantitation
of
lymphocyte
proliferative
response
to allogeneic
5.
analysis.
patients
to thank
in
to polyanions.
1959
Braunstein
cells
informadistribu-
or antigenic
stimulation.
Infrom
RNA
staining
is curto assess
the cytostatic
efin
2.
DR.
of transformed
metais ex-
cell
Bradley
bound
45:944,
counterstaining
agents,
wish
assistance
Traganos
(often
The
which
authors
her
LITERATURE
1.
permeability
of peripheral
cytes
to mitogenic
formation
obtained
rently
being
used
fects
used.
and
DNA
in
simple,
fast
cell systems
channels,
be obtained
from
response
fixation
manuscript.
staining
or a single
fluorescence)
various
specimens
the
differentia-
a two-step
pH 3.0,
The
for
procedures
have
been
analyses
of tissue
culture
by
P1, mithramycin,
to cell
ACKNOWLEDGMENT
is de-
fluorescence
ratios
may
and
bromide,
alternative
not
DNA
independent
and cell cycle
offering
(ethidium
an
information
cell
(green
dyes
reacting
activity
(red
fluorescence)
the addition
of pulse
width
thereby
analysis
does
of DNA
makes
identification
lines
treatment.
are required.
with
AO
circuits
on both
to cytoplasmic
23)
tonic
or centrifugations
provides
levels
one
as
membrane
system
and
ploidy
transcriptional
the other.
suring
nuclear
for
cell loss)
associated
in
unnecessary
tion
as to
etc.)
scale.
no washings
resulting
chromasia
other
3.0-4.0
55
ORANGE
of
4.0 should
be
of RNA
AO offers
a
a variety
of
microculture
ACRIDINE
in
stainable
at
USING
denaturation
when
etc.)
CELLS
be performed
it with
activity,
treatment
Simultaneous
even
pH
extent
in cases
quantity
unfixed
means
at
the
IN
Z, Moore
Z,
‘I’raganos
Lymphocyte
analysis.
Proc
F, Sharpless
T,
stimulation.
A rapid
Natl
Acad
Sci
US,
1976
10. Darzynkiewicz
Z,
Traganos
F, Sharpless
T,
Friend
C, Melamed
MR:
Nuclear
chromatin
changes
during erythroid
differentiation
of Friend
virus-induced
leukemic
cells. Exp Cell Res, 99:
301, 1976
11. Dukes
CD, Parsons
JL, Stephen
CAL:
Use of
acridine
orange
in lymphocyte
transformation
test. Proc Soc Exp Biol Med 131:1168,
1969
12. Kapaln
J, Shope TC, Peterson
WA Jr: EpsteinBarr-virus-negative
human
malignant
T cell
Lines. J Exp Med 139:1070,
1974
13.
Krishan
A:
of mammalian
staining.
Rapid
J Cell
cell
flow cytofluorometric
cycle
by propidium
Biol 66:188,
1975
analysis
iodide
56
TRAGANOS
14. LePecq
JB: Use of ethidium
bromide
for separation and determination
of nucleic
acids of various
conformational
forms and measurement
of their
associated
enzymes.
Methods
Biochem
Anal
20:41, 1971
15. LePecq
JB, Paoletti
C: A fluorescent
complex
between ethidium
bromide
and nucleic
acids.
J
Mol Biol 27:87,
1967
16. Lerman
LS: The structure of the DNA
acridine
complex.
Proc Natl Acad Sci US 49:94,
1963
17. Melamed
MR,
Adams
LR,
Traganos
F,
Kamentsky
LA: Blood Granulocyte
staining
with
acridine
orange.
Changes
with infection.
J Histochem
Cytochem
22:526, 1974
18. Melamed
MR, Kamentsky
LA: Automated
cytology. Internatl
Rev Pathol
14:205, 1975
19.
Ralph
P: Retention
of lymphoid
characteristics
by myeloma
and theta
lymphomas.
J Immun
110:1470,
1973
20. Rigler R Jr: Microfluorometric
characterization
of
intracellular
acridine
1,1966
nucleic
orange.
Acta
acids
Physiol
and
nucleoproteins
Scand
67:suppl
by
267,
ET
21.
22.
23.
AL.
Sharpless
TK, Melamed
MR: Estimation
of cell
size from pulse
shape
in flow cytofluorometry.
J Histochem
Cytochem
24:257,
1976
Steinkamp
JA, Crissman
HA: Automated
analysis of deoxyribonucleic
acid, protein
and nuclear
to cytoplasmic
relationships
in tumor
cells and
gynecologic
specimens.
J Histochem
Cytochem
22:616,
1974
Steinkamp
JA,
Hansen
KM,
Crissman
HA:
Flow
microfluorometric
and lightscatter
measurement
of nuclear
and cytoplasmic
size in mammalian
cells.J Histochem Cytochem
24:292, 1976
24. Tal M: Metal
ions and ribosomal
conformation.
Biochem
Biophys
Acta 95:76, 1969
25. Traganos
F, Darzynkiewicz
Z. Sharpless
T,
Melamed
MR: Denaturation
of deoxyribonucleic
acid in situ. Effect of formaldehyde.
J Histochem
Cytochem
23:431,
1975
26. Traganos
F, Darzynkiewicz
Z, Sharpless T,
Melamed
MR: Cytofluorometric
studies
on conformation
of nucleic
acids in situ. I. Restriction
of
acridine
orange binding
by chromatin
proteins.
J
Histochem
Cytochem
24:40, 1976