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A Method for Isolation of Nuclei from Cells of the Walker 256
Carcinosarcoma*
HARRIS BUSCH, WESLEY C. STARBUCK,t AND JOSEPH R. DAVISJ~
(Department of Pharmacology, University of Illinois College of
Medicine, Chicago 12, Ill.)
Recent reports from this laboratory (1, ~) have indicated the
desirability of a method for the preparation of nuclei free from
cytoplasmic con- tamination which would be applicable to tumors as
well as other tissues. A variety of methods have been reported to
produce satisfactory preparations of nuclei of liver cells, but
Dounce has emphasized the difficulties in obtaining preparations
from tumor cells of nuclei which are not contaminated with a large
number of whole cells or cytoplasmic fractions (5). The serious
problems in isolation procedures requiring the use of organic
solvents have been discussed (1). The use of strongly acidic media
would increase Ithe possibility of losses of acid-soluble nuclear
proteins into which rAysine- U-C ~4 is rapidly incorporated (~).
Accordingly, a number of procedures involving the use of sucrose
media were tested, and the method of Chauveau et a/. (:3) was found
to be most useful when modifi- cations were made. The main
modifications of the procedure of Chauveau et al. were: (a)
definition of the limits of pestle clearance for removal of ad-
herent cytoplasm from the nucleus in the course of homogenization
in isotonic sucrose; (b) use of ~.0 M sucrose for resuspension of
the 600 • g precipi- tate prior to sedimentation in a force ]field
of 40,000 X g for :30 minutes. With optimal condi- tions, nuclei of
the Walker ~56 careinosareoma could be isolated which were
essentially free of adherent cytoplasm, whole cells, or cytoplasmic
granules. Following the injection of L-lysine-U-C TM into
tumor-bearing rats, histones were extracted from nuclei of tumor
cells obtained by this method and were found to have approximately
the same specific activities as the histones obtained by methods
used previously (~).
MATERIALS AND M E T H O D S / Animals and preparation of
tissues.--The tech-
nics of transplantation of the tumors, anesthesia,
* These studies were supported in part by grants from the
American Cancer~Society and the Jane Coffin Childs Fund for Medical
Research.
t Trainee of United States Public Health Service. :~Postdoctoral
Fellow of the American Cancer Society.
Received for publication February 12, 1959.
exsanguination, and initial treatment of the tissues were not
changed from those described previous- ly (~).
Homogenization.--For the purpose of this re- port, the pestle
clearance is defined as the difference between the maximum outer
diameter of the pestle and the minimum inner diameter of the
homogenizer tube. The maximum outer diameter of the pestle was
determined to _ 0.0005 inch by means of a micrometer caliper. The
minimum inner diameter of the tube was determined by means of a
telescoping gauge, ~-zl z inch, fitted with a 9-inch handle. 1 All
pestles and tubes used in this study were calibrated in this way
before use in the experiment.
The initial homogenization of the tissues was carried out in
isotonic sucrose (9' 1, v: w) with ten strokes of a Teflon| pestle
in a glass homogeniz- er tube. The pestle clearance for this step
was 1~ X 10 -8 inch. The speed of the homogenizer motor was 1~00
r.p.m.
Homogenization at defined pestle clearance.-- After the initial
homogenization, the sample was filtered through a l~0-mesh
stainless steel screen with the aid of gentle suction and then was
trans- ferred to one of a series of all-glass homogenizers (6) with
pestle clearances ranging from 0.5 to 7 X 10 -3 inch; the
suspension was then subjected to five up and down strokes of the
pestle. The ho- mogenate was transferred to a l~-ml, glass centri-
fuge tube and subjected to a force field of 600 X g for 10 minutes
(7) in an International refrigerated centrifuge at 5 ~ C. The
supernatant solution was decanted and discarded.
Centrifugation in hypertonic sucrose.--To the precipitate, 9
volumes of sucrose were added. In various experiments, the molarity
of sucrose ranged from 1.5 to ~.5 M. After a few minutes, most of
the tissue rose to the top, leaving a small layer of packed red
cells at the bottom of the cen- trifuge tube. The homogenate and
sucrose were decanted into glass homogenizer tubes and re-
i The authors wish to express their appreciation to Mr. Bert
Gustafson, instrument-maker of the Department, for construction of
a handle of sufficient length for the telescoping gauge so that it
was possible to measure the internal diameters of the test tubes at
all levels of the tube.
684
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BUSCH et al.--Isolation of Nuclei from Walker Tumor 685
homogenized with glass pestles with a pestle clear- ance of 7-10
X 10 -3 inch. This preparation was centrifuged at 40,000 • g for 30
minutes in the Spinco Model L ultracentrifuge. The supernatant
solution was decanted from the clear jelly-like pel- let at the
bottom of the tube. To prevent aggre- gates of granules or the
supernatant solution from sliding into the pellet, the centrifuge
tubes were cut with scissors just above the pellet.
Phase microscopy.--Because of the reported dif- ficulties in the
detection of cytoplasmic aggregates adherent to the nuclei by means
of the light micro- scope (4), the phase microscope (A-O Spencer
Microstar) was used throughout this study. The bright-contrast oil
immersion lens was found to be particularly useful for detection of
cytoplasmic granules.
Purity of the preparations of nuclei.--In the preparations
obtained, differential counts were made of (a) nuclei completely
isolated from cyto- plasmic granules, (b) nuclei with adherent
cyto- plasm and granules, and (c) nuclei in intact cells. Several
counts of this type were made on each preparation in ten or more
fields to a total of 150 nuclei per count. The preparation was
repeated 7 times.
The purity of the preparation was also deter- mined by division
of the volume of the nuclei by the Volume of nuclei plus granules.
The nuclei and the granules were assumed to be spheres, and their
diameters were determined by means of a calibrat- ed ocular. The
diameter of the nuclei was found to average 18 g, while the average
diameter of the cytoplasmic granules was found to be 0.5 g.
RESULTS VARIABLES
Three variables were found to affect the yield and purity of the
nuclear preparation. In order, these were pestle clearance, number
of up-and- down strokes of the pestle, and molarity of the sucrose
used for the fractional sedimentation of the initial 600 X g
precipitate.
Pestle clearanee.--AU-glass homogenizers with pestle clearances
ranging from 0.5 X 10 -3 inch to 7 X 10- 3 inch were studied for
their effectiveness in removing the cytoplasm from the nuclei. As
indicated in Chart 1, the greatest ratio of isolated nuclei to
nuclei in intact cells was produced by homogenizers with a pestle
clearance of 3 X 10 -a inch.
As indicated in Chart 1, homogenizers with pestle clearances of
greater than 3 X 10- 3 inch produced a smaller ratio of isolated
nuclei to nuclei in cells. Homogenizers with smaller pestle
clearances resulted in significant destruction of the nuclei as
evidenced by the fact that relatively few
if any nuclei were found in each field even with a pestle
clearance of 1.5 X 10 -3 inch. With a pestle clearance of 0.5 X 10
-3 inch, essentially no nuclei were found, and a gelatinous mass
was noted. In a number of experiments with such tight homoge-
nizers, the initial 600 X g precipitate was greyish, as contrasted
with the pink color of the precipitate obtained after use of
homogenizers with pestle clearances of 3 X 10 -3 inch or
greater.
Strokes of the pestle.--Each preparation was sub- jected to five
or more up-and-down strokes of the homogenizer pestle through the
suspension. Im-
U3 J d
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ISOLATION OF TUMOR NUCLEI" PESTLE CLEARANGE
�9 , ,
2 4 6 PESTLE CLEARANCE (10 -3 INCH)
CHART 1.--Effect of pestle clearance on the ratio of isolated
nuclei to nuclei in cells. The greatest ratio was obtained with a
pestle clearance of 3 X 10 -a inch. In some of the preparations
with pestle clearances of 3 X 10 -s inch, essentially no whole
cells were found, but an occasional nucleus was noted with a small
halo of cytoplasm. When homogenizers with a pestle clearance of
less than 1.5 X 10 -3 inch were used, cells and nuclei were
completely broken. In most preparations a small percentage of
nuclei containing a little adherent cytoplasm could be found, and
the average number of these was 12 per cent at all the pestle
clearances studied. The data are average values for five
experiments at each point.
provement of the preparation resulted from a greater number of
strokes only when it was ob- served that the initial nuclear
preparation con- tained excess cytoplasm adherent to the nuclei.
When the preparation was subjected to more than 40 strokes of the
pestle through the suspension, the preparation was largely
denatured.
Molarity of sucrose for resuspension of the 600 X g
precipitate.--Molarities of sucrose ranging from 1.5 to ~.~ M were
tested in increments of 0.1 M. At molarities of sucrose ranging
from 1.5 to 1.8 M, the entire sample settled to the bottom of the
tube. Conversely, at molarities greater than ~.1 M, most of the
initial 600 X g precipitate remained sus- pended at the top of the
medium. In the interven- ing range of 1.9-~.1 M the nuclei settled
to the bot- tom of the tube, and highly purified preparations
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686 Cancer Research Vol. 19, July, 1959
could be obtained which were largely free of cyto- plasmic
granules. However, the yield at 3.1 M was less than that obtained
at ~.0 M; hence, the latter concentration was selected as the
standard con- centration for these experiments.
Effectiveness of the procedure.--From the studies above, the
following conditions were selected for isolation of nuclei of the
Walker tumor: (a) re- homogenization of the sample filtered through
the stainless steel screen (130 mesh) with an all-glass homogenizer
having a pestle clearance of 8 X 10 -3 inch; (b) five up-and-down
(c) fractional centrifugation precipitate in ~.0 M sucrose.
The effectiveness of this producing isolated nuclei is
strokes of the pestle; of the initial 600 X g
over-all procedure in noted in Figures 1-3.
Figure 1 is a photograph of a 600 X g precipitate obtained by
the Schneider procedure (7) with a homogenizer having a pestle
clearance of 7 X 10 -3 inch. I t can be seen that isolated nuclei
are rare, and whole cells are common. A number of the nuclei are
surrounded by cytoplasmic granules which are adherent to the
nucleus or are embedded in semicircular bits of cytoplasm. Figures
3 and 3 present the appearance of a microscopic field of nuclei
obtained by the method outlined above. 2 The few dark granules
interspersed among the nuclei are ground glass particles. Most of
the nuclei are completely free of adherent cytoplasmic
granules.
Glass.--In all preparations made in glass ho- mogenizers, highly
refractile particles were found which ranged in size from that of
small intracellu- lar granules to one fourth the diameter of the
nu- cleus. The amount of such particles markedly in- creased as the
pestle clearance diminished. Figure 3 indicates the appearance of
such particles when viewed through a bright-contrast oil immersion
lens. The nuclei in this field are very similar to those seen in
Figure 3, which is a photograph through a dark-contrast oil
immersion lens. When
2 I t was noted that a number of the nuclei of tumor cells had a
slight invagination of the nuclear membrane slightly off the center
of the nucleus. Because of this slight folding, some of these
nuclei appeared to be kidney bean-shaped.
homogenizers with a pestle clearance of 1.5 • 10 -3 inches or
less were used in control experiments without tissue, such
particles were found in large numbers. While the role of these very
small frag- ments of glass is not dear, it is possible that they
function to shear the cytoplasm off the nuclear membrane. When
Teflon| pestles were used to homogenize tissues in glass tubes,
many fewer par- titles were found, even with pestle clearances of
~.5 X 10 -3 inch. However, in such preparations essentially all the
nuclei either were inclosed in large cytoplasmic masses or were
found in whole cells. In occasional cases in which very few glass
fragments were found after homogenization in the all-glass pestles,
considerable cytoplasm was found adherent to the nuclei. In these
studies, then, there was a correlation between the amount of glass
present in the preparation and the effectiveness of the
homogenization in shearing the cytoplasm from the nucleus.
DISCUSSION From the data indicated above, it is apparent
that requirements for isolation of the nuclei of the Walker 356
carcinosarcoma include (a) an all-glass homogenizer with a pestle
clearance of 8 X 10 -s inch, (b) five up and down strokes of the
pestle (motor speed, 1~00 r.p.m.) and (c) recentrifugation of the
initial 600 X g precipitate in 3.0 M sucrose. The preparations of
nuclei varied in purity from 88 to 99 per cent when counts were
made of com- pletely isolated nuclei, nuclei with adherent cyto-
plasm, and nuclei in intact cells. Isolated nuclei comprised 99 per
cent of the total volume occupied by cellular particles in the best
preparations.
Limitations.--With this method, excellent prep- arations of
nuclei of Walker tumor and liver were obtained. However, the
preparations of nuclei of kidney and spleen were considerably ~more
con- taminated, even under the best conditions tested. I t should
be noted that the conditions required for isolation of good
preparations of the nuclei of the liver were not nearly so
stringent as those required for the tumor. For example, a Teflon|
pestle in a glass tube sufficed to produce a preparation in
leto. 1.--Nuclei and cells from the 600 g precipitate of the
Walker 256 tumor prepared by homogenization of the sample in an
all-glass homogenizer with a pestle clearance of 7 X 10 -8 inch.
There are a few whole cells with demarcated cell borders, some
nuclei with adherent cytoplasmic granules, and an occa- sional
nucleus which is free of cytoplasmic contamination. The field is
viewed through a bright-contrast oil immersion lens (X970) of the
phase microscope. In all photographs the cells and nuclei were
suspended in 0.9.5 M sucrose.
Fro. 2.--Nuclei isolated from the Walker 256 carcinosar-
coma by the method described in the text. The few dark gran-
ules noted in the preparation are glass particles. Differences in
the levels of the nuclei obscure some and brighten others. The
field is viewed through a dark-contrast oil immersion lens of the
phase microscope (X970).
I~G. 8.--Refractile bodies of ground glass between nuclei in a
field viewed through a bright-contrast oil immersion lens of the
phase microscope (• The nuclei in this field can be compared with
those of Figure 1, which are surrounded by cytoplasm and
granules.
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BuscrI et al.uIsoIation of Nuclei from Walker Tumor 687
which a large number of the nuclei of the liver were isolated
from the cytoplasm. When these prepara- tions were subjected to
centrifugation in the 2.0 M sucrose, nuclei were obtained in
moderately good yield, i.e., about 30--50 per cent of the nuclei
either in cells or with adherent cytoplasm and granules. I t
remains to be determined whether the ease of separation of nuclei
from cells is a biological char- acteristic of different
tissues.
Confirmation of data on uptake of L-lysine-U-C 14 into the
histones.--In a recent study (2) data were obtained which indicated
that the uptake of iso- tope into the histones of the Walker and
Jensen tumor exceeded that of most other tissues when radioactive
lysine was used as the precursor. Fol- lowing the development of
the present method, this problem has been further investigated, and
it has been found that the specific activities of the histones
obtained from nuclei of the liver and of the Walker tumor do not
vary significantly, i.e., 5 per cent or less, from the data
reported previ- ously. With the present method, the total weight of
histones recovered from the nuclei of the cells of the Walker tumor
was 95-100 per cent of the total histones obtained in previous
experiments (2), while the total weight of histones recovered from
the liver was 40-53 per cent of the total previously obtained.
SUMMARY 1. To obtain isolated nuclei of the Walker 256
carcinosarcoma, samples of the tumor were sub- jected to initial
homogenization in 0.25 M sucrose in a glass homogenizer tube fitted
loosely with a
Teflon| pestle and having a pestle clearance of 9-12 X 10 -a
inch. The homogenate was re- homogenized in an all-glass
homogenizer with a pestle clearance of 3 X 10 -s inch, and then
cen- trifuged at 600 X g for 10 minutes. The precipi- tated nuclei
and debris were resuspended in 2.0 M sucrose and recentrifuged at
40,000 X g for 30 minutes.
2. The nuclei were obtained in high yield with a purity of 99
per cent by volume and 88-99 per cent by differential count of
isolated nuclei, nuclei with fragments of adherent cytoplasm, and
nuclei in intact cells.
REFERENCES
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Tissues: A Pw~/ew. Cancer Research, 18:1~41-56, 1958.
~. BUSCH, H.; DAvis, J. R.; and A~m~RSON, D. C. Labeling of
Histones and Other Nuclear Proteins with L-Lysine-U-C 14 in Tissues
of Tumor-bearing Rats. Cancer Research, 18: 916-~6, 1958.
3. CHAUVEAU, J.; Mou~ , Y.; and ROWLU~R, C. H. Isolation of Pure
and Unaltered Liver Nuclei, Morphology and Bio- chemical
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4. DAviSON, P. F., and MERCER, E. H. Electron Microscopy of Cell
Nuclei Isolated in Aqueous Media. Exper. Cell Re- search, 11:
~7-39, 1956.
5. DOUNCE, A. L. The Isolation and Composition of Cell Nu- clei
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7. SCHNEIDER, W. C. Intracellular Distribution of Enzymes. III.
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176: ~59-66, 1948.
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~ ! i i~!i~ !�84184184184184 ~
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1959;19:684. Cancer Res Harris Busch, Wesley C. Starbuck and
Joseph R. Davis CarcinosarcomaA Method for Isolation of Nuclei from
Cells of the Walker 256
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