Vol.
183,
March
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
16, 1992
Pages
Propidium
iodide stainhg
Tessa Crompton*,
+Institute
Received
cormla~ with the extent of DNA degradation in isolated nuclei
Manuel
*Ludwig
Institute
C. Peitsch+,
23,
H. Rohson MacDonald*
for Cancer Research,
of Biochemistry,
January
532437
University
and Jiirg Tschopp+
1066-Epalinges,
of Lausanne,
Switzerland
1066-Epalinges,
Switzerland
1992
SUMMARY Gradual degradation of internucleosomal DNA is a hallmark of apoptosis and can be simulated by incubating isolated thymocyte nuclei in the presence of 5 mM Mg2+ and 5 mM Ca2+ at 37°C. Staining of nuclei with the DNA binding fluorescent dye propidium iodide (PI) showed that intensity of fluoresence correlated with the extent of DNA degradation. PI fluorescence was increased in the presence of DNase I. Thus it seems that the cleavage of chromatin DNA by DNase 1 or by the endogenous enzyme increases the accessibility of DNA for the dye. No increase of fluorescence was observed in the presence of the known inhibitors of the endogeneous endonuclease: Zn2+ and EGTA. However, the presence of Zn 2+ led to decreased staining of the nuclei by PI and caused a shift in the scatter profile of the nuclei, suggesting that a conformational change of chromatin is induced by this ion. This correlation between intensity of PI staining and DNA degradation should be useful to compare endogenous nuclease levels in lymphocyte populations. 0 1992 Academicpress, 1°C.
Apoptosis developmentally in the murine
has been implicated programmed
as a mechanism
system (1) including
thymus (2-4). DNA degradation,
Ca2+ and Mg2+
dependent
endogenous
clonal deletion
purportedly
endonuclease
To date the measurement
qualitative
of autoreactive
caused by the activation
such as agarose gel electrophoresis of isolated
PI is an intercalating cytometry
for quantative
addition “gate”
the exclusion when analyzing
been recently thymocytes
exploited undergoing
intemucleosomal thymocyte 0006-291X/92 Copyright All rights
cells of a
has relied on
of DNA prepared
from dying
using propidium
nuclei and FACS analysis. DNA-binding
dye that has been widely
cell cycle analysis
of fixed
of PI by living
cells is frequently
heterogeneous
populations.
used in flow
cells and isolated
nuclei.
used to establish
The latter
property
apoptosis
DNA degradation
in response to autoantigens correlates
nuclei.
$1.50 Inc. reserved.
532
quantatively
(7).
In
a viable cell of PI has also
to define the late stages of cell death in populations
0 1992 by Academic Press, oj~ reproduction in any form
of
as a defining
of such DNA fragmentation
eels. Here we present a rapid method to quantify this DNA degradation, iodide (PI) staining
in a variety
(5,6), is regarded
feature of apoptosis. methods
of cell death
of immature
Here we show that
with PI staining
in isolated
Vol.
BIOCHEMICAL
183, No. 2, 1992
Buffers and solutions.
7.5 containing buffer:
40 mM Tris-acetate
was slightly
0.5 mM spermidin
isolation.
pH 8.0 containing The procedure
modified
and applied
Dounce homogeniser
iodide (PI)(Sigma,
for nuclei isolation
The cell suspension
removed with a glass pipette.
5 ml of serum-free
medium
(Dulbecco-MEM)
ice-cold buffer B containing by 5-10 strokes volume white
in a Dounce homogeniser. pellet
same 30 % sucrose concentration
cushion.
of 108 nuclei/ml
DNA degradation
(Dulbecco-MEM)
in 3 ml
and disrupted
were underlayered
with
an equal
for 10 min at 1OOOg. The
in 3 ml buffer A and recentrifuged was resuspended
and kept on ice until used (within
in nuclei.
was performed
1 ml / lo8 thymocytes)
pellet
using a
The cells were resuspended
They
The final
(8)
Cells were washed 2 times in
30 % sucrose and centrifuged
was resuspended
and Burgoyne
and the last centrifugation
10 % sucrose.
0.05 % NP-40 (approx.
of buffer A containing nuclear
TAE
was put on ice for 10 min and
material
2 ml of buffer A containing
of Hewish
medium
coarse particulated through
St-Louis).
pH
A thymus was collected from a 4-6 weeks
in 5 ml serum-free
(pestel B).
Buffer
Buffer C: 20 mM Tris-acetate
1 mM EDTA
as follows:
old Balb/c mouse and disrupted
60 mM KCI, 15
and 14 mM b-mercaptoethanol.
5 mM EDTA and 5 mM EGTA.
10 mM Mg2+ and 50 ug/ml propidium
Nuclei
RESEARCH COMMUNICATIONS
Buffer A: 15 mM Hepes (pH 7.5) containing
mM NaCl, 0.15 mM spermin, B: buffer A containing
AND BIOPHYSICAL
lo5 nuclei were incubated
24
through
in buffer
the
A at a
hrs).
in a final volume of 10 ul
in the presence of 5 mM Mg2+ and 5 mM Ca2+ at 37°C for 0, 15, 30, 45 and 60 min; the reaction
was stopped by the addition
of 0.5 ml ice-cold buffer C and placing the samples
on ice. In the absence of Ca 2+, Mg2+ was added at a concentration order to keep the same concentration The inhibitory addition
of divalent
cations) in presence
effect of Zn2+ was tested at final concentrations
to the normal
concentrations
of 10 mM Mg2+ (in
of Mg 2+ and Ca2+.
of 5 mM EGTA.
of 0.1, 1 and 10 mM in
Potential
maximal
iodide staining
was assessed by adding 0.1 mglml of bovine pancreatic
to the reaction
mixture
propidium
DNase I (Sigma)
in presence of 5 mM Mg2+ and 5 mM Ca2+, and assayed as the
other samples. Agarose reaction
gel electrophoresis
was stopped
alcohol (50/49/l
of DNA.
by the addition
At the end of the incubation
of three
by volumes) and mixing.
volumes
After centrifugation
After a migration
of 30-60 min at 5 Volts/cm,
the
of phenol/choloform/isoamylat 12000 x g for 5 min, the
samples were loaded onto a 1.8 % agarose gel in TAE buffer containing bromide.
period,
photographs
1 ug/ml ethidium were taken on a HV
transiluminator. Flow incubated Facscan
cytometry.
For assessment
of DNA fragmentation,
at 4OC for 15 min in buffer C containing (Becton
Dickinson).
Fluorescence
scale. 533
isolated
nuclei
50 ug/ml PI prior to analysis
was recorded
and analyzed
were on a
on a linear
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
RESULTS Propidium
nuclease
containing
Mg2+
various
was induced
RESEARCH
COMMUNICATIONS
AND DISCUSSION
iodide (PI) staining
endogenous
RIOPHYSICAL
as a measure of DNA degradation.
by incubation
of isolated
thymocyte
and Ca2+ (9) and the extent of DNA degradation
time points by gel electrophoresis
of extracted
Activation nuclei
staining
(Fig.
increased
with
time.
Thus,
This
degradation. correlation
Nuclei
forward
propidium incubated
DNA (Fig. 1A). At the same time
of PI staining
mean fluorescence is supported iodide
degradation
is an index by recent
uptake
1A) and
of the extent
of DNA
demonstrating
nuclease
the
treatment
and DNA degradation,
Again there was a clear relationship
we compared
incubated
with
between intensity
a maximum
mean fluorescence
between
gel electrophoresis
or without
of staining
As expected, DNA cleavage occurred more rapidly
DNase I reaching
DNase
and
I (Fig.
2).
and extent of in the presence of
after 45 minutes.
The subsequent
decline was probably the result of some DNA loss from nuclei, as at later time points some debris were observed on the light scatter (foward scatter versus 90” scatter) plot.
12345
v)
=
Fiaure
1. Propidium iodide staining correlates with the extent of nuclear DNA degradation. 105 thymocyte nuclei were incubated in presence of 5 mM Mg2+ and 5 mM Ca2+ for various periods. Samples were diluted 50-fold in a buffer containing 10 mM Mg2+ and 50 mg/ml propidium iodide and incubated for 10 min on ice before flow cytometry. A) agarose gel analysis of DNA extracted from the samples at times 0,15,30, and 60 min (lanes l-4), and molecular weight markers: 1608, 1212, 619, 587, 517,396, 317,219 and 132 base pairs from top to bottom (lane 5), B) overlap of propidium iodide fluorescence histograms at times 0, 15,30 and 60 min (the numbers correspond to the samples in lanes 1-4 in A). 534
of
and Ca2+ for 0 to 60 min show
To verify the correlation
increased by DNase I.
of
(Fig.
work
and restriction
in the presence of Mg2+
Fats analysis of isolated thymocyte nuclei degradation.
of DNA
Intensity
versus 90” scatter plots.
DNA degradation intensity
to the extent
observation
between
nuclei (10). similar
1B) correlated
in buffer
was assessed at
points, isolated nuclei were stained with PI and analyzed by flow cytometry. PI
of
Vol.
183,
No.
2, 1992
BIOCHEMICAL
8! 8 5 E al E
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
180
160
x 140 E 2 EQ 120 2 0. s @ 100 5 0
40 20 Time (min)
60
Fimre 2, The increase of propidium iodide staining by thymocyte nuclei is accelerated by DNaseI. A) IO5 thymocyte various periods Mean propidium
nuclei iodide
DNA degradation increase in PI staining known
inhibitors
were incubated
in presence
of 5 mM
Mg2+
and 5 mM
in the presence (squares) and absence (circles) of 0.1 mg/ml fluorescences
is inhibited
are plotted
time.
by Zn2+ and EGTA.
in isolated thymocyte
of thymocyte
versus
Zn2+
and EGTA inhibited
nuclei (Fig. 3A). These reagents
endonuclease
activity (9). Gel electrophoresis
prepared
from the same samples showed that DNA degradation
inhibited
(Fig. 3B).
packing which might explain
the inhibitory
values were actually
and nuclease activity appreared
to be totally abrogated
are of DNA
in the presence of 10
mM Zn2+ we observed an increase in 90” scatter (Fig 3C to E) suggesting this case the mean fluorescence
the
had been similarly
In the presence of 1 mM Zn2+, and even more pronounced chromatin
Ca2+ for DNase I.
some change in
action of Zn2+ on the enzyme. reduced (as compared
In
to controls),
(Fig. 3A and B).
CONCLUSION In conclusion
it is widely accepted that intemucleosomal
useful marker for apoptotic here demonstrate nuclei followed Although presumably
that a rapid and simple method of PI staining by FACS analysis allows quantitation
the precise mechanism is related to increased
underlying
populations
at different
of isolated thymocyte
of such DNA degradation.
this phonomenon
accessibility
dye. This method should enable us to quantitate lymphocyte
DNA degradation
is a
cell death in a variety of model systems. The data presented
of degraded endogenous
is not known, it DNA to the intercalating endonulease
stages of their development.
535
levels in
Vol.
183, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
6 12345
E 60-j-----J ii 0 20
40
Time
60
(min)
FSC ( A.U. )
SSC ( A.U. ) Fieure 3. Influence of divalent cations on propidium iodide staining of thymocyte nuclei. A) 105 thymocyte nuclei were incubated in presence of 1) 5 mM Mg2+ and 5 mM Ca2+ (open squares); 2) 5 mM Mg2’, 5 mM Ca2+ and 0.1 mM Zn2+ (closed circles); 3) 5 mM Mg2+, 5 mM Ca2+ and 1 mM Zn2+ (closed circles with dashed line); 4) 5 mM Mg2+, 5 mM Ca2+ and 10 mM Zn2+ (open circles) and finally 5) 10 mM Mg2+ and 5 mM EGTA (closed squares). Samples were treated as stated under Material and Methods. B) After 45 min of incubation, DNA was extracted from all samples and were analysed by agarose gel electrophoresis. Numbers on corresponding data points and gel lanes are identical. FSC versus SSC plot of tbymocyte nuclei in absence (C) and presence (D) of 10 mM Zn2+. E) overlap of the SSC distribution shown in C and D. Some doublets, or nuclei in G2 are present.
ACKNOWLEDGMENTS We thank the Wellcome
P. Zaech Trust
and
C. Knabenhans
and MC.
Peitsch
for cell sorting. was
supported
536
T. Crompton
by the Swiss
was Cancer
supported League.
by
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
1. Duvall, E. and Wyllie, A. H. (1986) Immunol. Today 2. Shi, Y., Sahai, B. M. and Green, D. R. (1989) Nature 3. MacDonald,
H. R. and Lees, R. K (1990) Nature
4. Smith, C. A., Williams, (1990) Nature
G. T., Kingston,
RESEARCH
COMMUNICATIONS
7, 115-119. 339, 625--626.
343,642-644.
R., Jenkinson,
E. J. and Owen, J. J. T.
38’7,181-184.
5. Wyllie, A. H. (1980) Nature
234,555-556.
6. Cohen, J. J., and Duke, R. C. (1984) J. Immunol. 7. Swat, W., Ignatowicz,
132,38-42.
L., von Boehmer, H. and Risielow,
P. (1991) Nature
351,
Xx-156. 8. Hewish, D. R. and Burgoyne,
L. A. (1973) (1973)
Biochem. Eiophys. Res. Commurz.
52,504510. 9. Vanderbilt,
J. N., Bloom, K. S. and Anderson,
J. N. (1982) J. Bioi. Chem. 257,
13009-13017. 10. Prosperi,
E., Giangare,
C. and Bottiroli,
G. (1991) Cytometry
537
12,323-329.
183,
March
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
16, 1992
Pages
Propidium
iodide stainhg
Tessa Crompton*,
+Institute
Received
cormla~ with the extent of DNA degradation in isolated nuclei
Manuel
*Ludwig
Institute
C. Peitsch+,
23,
H. Rohson MacDonald*
for Cancer Research,
of Biochemistry,
January
532437
University
and Jiirg Tschopp+
1066-Epalinges,
of Lausanne,
Switzerland
1066-Epalinges,
Switzerland
1992
SUMMARY Gradual degradation of internucleosomal DNA is a hallmark of apoptosis and can be simulated by incubating isolated thymocyte nuclei in the presence of 5 mM Mg2+ and 5 mM Ca2+ at 37°C. Staining of nuclei with the DNA binding fluorescent dye propidium iodide (PI) showed that intensity of fluoresence correlated with the extent of DNA degradation. PI fluorescence was increased in the presence of DNase I. Thus it seems that the cleavage of chromatin DNA by DNase 1 or by the endogenous enzyme increases the accessibility of DNA for the dye. No increase of fluorescence was observed in the presence of the known inhibitors of the endogeneous endonuclease: Zn2+ and EGTA. However, the presence of Zn 2+ led to decreased staining of the nuclei by PI and caused a shift in the scatter profile of the nuclei, suggesting that a conformational change of chromatin is induced by this ion. This correlation between intensity of PI staining and DNA degradation should be useful to compare endogenous nuclease levels in lymphocyte populations. 0 1992 Academicpress, 1°C.
Apoptosis developmentally in the murine
has been implicated programmed
as a mechanism
system (1) including
thymus (2-4). DNA degradation,
Ca2+ and Mg2+
dependent
endogenous
clonal deletion
purportedly
endonuclease
To date the measurement
qualitative
of autoreactive
caused by the activation
such as agarose gel electrophoresis of isolated
PI is an intercalating cytometry
for quantative
addition “gate”
the exclusion when analyzing
been recently thymocytes
exploited undergoing
intemucleosomal thymocyte 0006-291X/92 Copyright All rights
cells of a
has relied on
of DNA prepared
from dying
using propidium
nuclei and FACS analysis. DNA-binding
dye that has been widely
cell cycle analysis
of fixed
of PI by living
cells is frequently
heterogeneous
populations.
used in flow
cells and isolated
nuclei.
used to establish
The latter
property
apoptosis
DNA degradation
in response to autoantigens correlates
nuclei.
$1.50 Inc. reserved.
532
quantatively
(7).
In
a viable cell of PI has also
to define the late stages of cell death in populations
0 1992 by Academic Press, oj~ reproduction in any form
of
as a defining
of such DNA fragmentation
eels. Here we present a rapid method to quantify this DNA degradation, iodide (PI) staining
in a variety
(5,6), is regarded
feature of apoptosis. methods
of cell death
of immature
Here we show that
with PI staining
in isolated
Vol.
BIOCHEMICAL
183, No. 2, 1992
Buffers and solutions.
7.5 containing buffer:
40 mM Tris-acetate
was slightly
0.5 mM spermidin
isolation.
pH 8.0 containing The procedure
modified
and applied
Dounce homogeniser
iodide (PI)(Sigma,
for nuclei isolation
The cell suspension
removed with a glass pipette.
5 ml of serum-free
medium
(Dulbecco-MEM)
ice-cold buffer B containing by 5-10 strokes volume white
in a Dounce homogeniser. pellet
same 30 % sucrose concentration
cushion.
of 108 nuclei/ml
DNA degradation
(Dulbecco-MEM)
in 3 ml
and disrupted
were underlayered
with
an equal
for 10 min at 1OOOg. The
in 3 ml buffer A and recentrifuged was resuspended
and kept on ice until used (within
in nuclei.
was performed
1 ml / lo8 thymocytes)
pellet
using a
The cells were resuspended
They
The final
(8)
Cells were washed 2 times in
30 % sucrose and centrifuged
was resuspended
and Burgoyne
and the last centrifugation
10 % sucrose.
0.05 % NP-40 (approx.
of buffer A containing nuclear
TAE
was put on ice for 10 min and
material
2 ml of buffer A containing
of Hewish
medium
coarse particulated through
St-Louis).
pH
A thymus was collected from a 4-6 weeks
in 5 ml serum-free
(pestel B).
Buffer
Buffer C: 20 mM Tris-acetate
1 mM EDTA
as follows:
old Balb/c mouse and disrupted
60 mM KCI, 15
and 14 mM b-mercaptoethanol.
5 mM EDTA and 5 mM EGTA.
10 mM Mg2+ and 50 ug/ml propidium
Nuclei
RESEARCH COMMUNICATIONS
Buffer A: 15 mM Hepes (pH 7.5) containing
mM NaCl, 0.15 mM spermin, B: buffer A containing
AND BIOPHYSICAL
lo5 nuclei were incubated
24
through
in buffer
the
A at a
hrs).
in a final volume of 10 ul
in the presence of 5 mM Mg2+ and 5 mM Ca2+ at 37°C for 0, 15, 30, 45 and 60 min; the reaction
was stopped by the addition
of 0.5 ml ice-cold buffer C and placing the samples
on ice. In the absence of Ca 2+, Mg2+ was added at a concentration order to keep the same concentration The inhibitory addition
of divalent
cations) in presence
effect of Zn2+ was tested at final concentrations
to the normal
concentrations
of 10 mM Mg2+ (in
of Mg 2+ and Ca2+.
of 5 mM EGTA.
of 0.1, 1 and 10 mM in
Potential
maximal
iodide staining
was assessed by adding 0.1 mglml of bovine pancreatic
to the reaction
mixture
propidium
DNase I (Sigma)
in presence of 5 mM Mg2+ and 5 mM Ca2+, and assayed as the
other samples. Agarose reaction
gel electrophoresis
was stopped
alcohol (50/49/l
of DNA.
by the addition
At the end of the incubation
of three
by volumes) and mixing.
volumes
After centrifugation
After a migration
of 30-60 min at 5 Volts/cm,
the
of phenol/choloform/isoamylat 12000 x g for 5 min, the
samples were loaded onto a 1.8 % agarose gel in TAE buffer containing bromide.
period,
photographs
1 ug/ml ethidium were taken on a HV
transiluminator. Flow incubated Facscan
cytometry.
For assessment
of DNA fragmentation,
at 4OC for 15 min in buffer C containing (Becton
Dickinson).
Fluorescence
scale. 533
isolated
nuclei
50 ug/ml PI prior to analysis
was recorded
and analyzed
were on a
on a linear
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
RESULTS Propidium
nuclease
containing
Mg2+
various
was induced
RESEARCH
COMMUNICATIONS
AND DISCUSSION
iodide (PI) staining
endogenous
RIOPHYSICAL
as a measure of DNA degradation.
by incubation
of isolated
thymocyte
and Ca2+ (9) and the extent of DNA degradation
time points by gel electrophoresis
of extracted
Activation nuclei
staining
(Fig.
increased
with
time.
Thus,
This
degradation. correlation
Nuclei
forward
propidium incubated
DNA (Fig. 1A). At the same time
of PI staining
mean fluorescence is supported iodide
degradation
is an index by recent
uptake
1A) and
of the extent
of DNA
demonstrating
nuclease
the
treatment
and DNA degradation,
Again there was a clear relationship
we compared
incubated
with
between intensity
a maximum
mean fluorescence
between
gel electrophoresis
or without
of staining
As expected, DNA cleavage occurred more rapidly
DNase I reaching
DNase
and
I (Fig.
2).
and extent of in the presence of
after 45 minutes.
The subsequent
decline was probably the result of some DNA loss from nuclei, as at later time points some debris were observed on the light scatter (foward scatter versus 90” scatter) plot.
12345
v)
=
Fiaure
1. Propidium iodide staining correlates with the extent of nuclear DNA degradation. 105 thymocyte nuclei were incubated in presence of 5 mM Mg2+ and 5 mM Ca2+ for various periods. Samples were diluted 50-fold in a buffer containing 10 mM Mg2+ and 50 mg/ml propidium iodide and incubated for 10 min on ice before flow cytometry. A) agarose gel analysis of DNA extracted from the samples at times 0,15,30, and 60 min (lanes l-4), and molecular weight markers: 1608, 1212, 619, 587, 517,396, 317,219 and 132 base pairs from top to bottom (lane 5), B) overlap of propidium iodide fluorescence histograms at times 0, 15,30 and 60 min (the numbers correspond to the samples in lanes 1-4 in A). 534
of
and Ca2+ for 0 to 60 min show
To verify the correlation
increased by DNase I.
of
(Fig.
work
and restriction
in the presence of Mg2+
Fats analysis of isolated thymocyte nuclei degradation.
of DNA
Intensity
versus 90” scatter plots.
DNA degradation intensity
to the extent
observation
between
nuclei (10). similar
1B) correlated
in buffer
was assessed at
points, isolated nuclei were stained with PI and analyzed by flow cytometry. PI
of
Vol.
183,
No.
2, 1992
BIOCHEMICAL
8! 8 5 E al E
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
180
160
x 140 E 2 EQ 120 2 0. s @ 100 5 0
40 20 Time (min)
60
Fimre 2, The increase of propidium iodide staining by thymocyte nuclei is accelerated by DNaseI. A) IO5 thymocyte various periods Mean propidium
nuclei iodide
DNA degradation increase in PI staining known
inhibitors
were incubated
in presence
of 5 mM
Mg2+
and 5 mM
in the presence (squares) and absence (circles) of 0.1 mg/ml fluorescences
is inhibited
are plotted
time.
by Zn2+ and EGTA.
in isolated thymocyte
of thymocyte
versus
Zn2+
and EGTA inhibited
nuclei (Fig. 3A). These reagents
endonuclease
activity (9). Gel electrophoresis
prepared
from the same samples showed that DNA degradation
inhibited
(Fig. 3B).
packing which might explain
the inhibitory
values were actually
and nuclease activity appreared
to be totally abrogated
are of DNA
in the presence of 10
mM Zn2+ we observed an increase in 90” scatter (Fig 3C to E) suggesting this case the mean fluorescence
the
had been similarly
In the presence of 1 mM Zn2+, and even more pronounced chromatin
Ca2+ for DNase I.
some change in
action of Zn2+ on the enzyme. reduced (as compared
In
to controls),
(Fig. 3A and B).
CONCLUSION In conclusion
it is widely accepted that intemucleosomal
useful marker for apoptotic here demonstrate nuclei followed Although presumably
that a rapid and simple method of PI staining by FACS analysis allows quantitation
the precise mechanism is related to increased
underlying
populations
at different
of isolated thymocyte
of such DNA degradation.
this phonomenon
accessibility
dye. This method should enable us to quantitate lymphocyte
DNA degradation
is a
cell death in a variety of model systems. The data presented
of degraded endogenous
is not known, it DNA to the intercalating endonulease
stages of their development.
535
levels in
Vol.
183, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
6 12345
E 60-j-----J ii 0 20
40
Time
60
(min)
FSC ( A.U. )
SSC ( A.U. ) Fieure 3. Influence of divalent cations on propidium iodide staining of thymocyte nuclei. A) 105 thymocyte nuclei were incubated in presence of 1) 5 mM Mg2+ and 5 mM Ca2+ (open squares); 2) 5 mM Mg2’, 5 mM Ca2+ and 0.1 mM Zn2+ (closed circles); 3) 5 mM Mg2+, 5 mM Ca2+ and 1 mM Zn2+ (closed circles with dashed line); 4) 5 mM Mg2+, 5 mM Ca2+ and 10 mM Zn2+ (open circles) and finally 5) 10 mM Mg2+ and 5 mM EGTA (closed squares). Samples were treated as stated under Material and Methods. B) After 45 min of incubation, DNA was extracted from all samples and were analysed by agarose gel electrophoresis. Numbers on corresponding data points and gel lanes are identical. FSC versus SSC plot of tbymocyte nuclei in absence (C) and presence (D) of 10 mM Zn2+. E) overlap of the SSC distribution shown in C and D. Some doublets, or nuclei in G2 are present.
ACKNOWLEDGMENTS We thank the Wellcome
P. Zaech Trust
and
C. Knabenhans
and MC.
Peitsch
for cell sorting. was
supported
536
T. Crompton
by the Swiss
was Cancer
supported League.
by
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
1. Duvall, E. and Wyllie, A. H. (1986) Immunol. Today 2. Shi, Y., Sahai, B. M. and Green, D. R. (1989) Nature 3. MacDonald,
H. R. and Lees, R. K (1990) Nature
4. Smith, C. A., Williams, (1990) Nature
G. T., Kingston,
RESEARCH
COMMUNICATIONS
7, 115-119. 339, 625--626.
343,642-644.
R., Jenkinson,
E. J. and Owen, J. J. T.
38’7,181-184.
5. Wyllie, A. H. (1980) Nature
234,555-556.
6. Cohen, J. J., and Duke, R. C. (1984) J. Immunol. 7. Swat, W., Ignatowicz,
132,38-42.
L., von Boehmer, H. and Risielow,
P. (1991) Nature
351,
Xx-156. 8. Hewish, D. R. and Burgoyne,
L. A. (1973) (1973)
Biochem. Eiophys. Res. Commurz.
52,504510. 9. Vanderbilt,
J. N., Bloom, K. S. and Anderson,
J. N. (1982) J. Bioi. Chem. 257,
13009-13017. 10. Prosperi,
E., Giangare,
C. and Bottiroli,
G. (1991) Cytometry
537
12,323-329.
