A General and Reliable Method for
Obtaining High-Yield Metaphasic
Preparations from Adherent Cell Lines:
Rapid Verification of Cell Chromosomal
studies are becoming more and more frequent
in numerous domains of cell biology. For such
purposes, a great variety of cell lines have been established
either from normal tissues or, more frequently,
from tumours. Very few of these cells present a normal
karyotype: they are generally hyperdiploid and often
contain rearranged chromosomes. Moreover, depending
on the length of time in culture and on the culture
conditions, populations of these cell lines can change
and drift. Therefore, some general culture rules have
to be respected (Ian Freshney, 1987), and it is highly
recommended to avoid working with cells maintained
in culture for a large number of generations.
Because of possible drift, cell lines need to be followed
and controlled, with one fundamental control
being the stability of their chromosomal content.
Unfortunately, such control is not systematic. Moreover,
in the case of new cell lines, it is frequent that
their phenotype is described in detail with little or no
information on their karyotype. It should be noted that
even if karyotyping methods have long been described
in the literature (for the history of human cytogenetics,
see Jeening Lawce and Brown, 1997), it is not
always easy to obtain metaphasic preparations from
some cell lines. We were confronted with such a situation
for some hybrid lines, in particular polarized rat
hepatoma-human fibroblast WIF clones (Cassio et al.
1991; Shanks et al.
, 1994). This article describes a new
method for obtaining, at a high yield, metaphasic
preparations from delicate cell lines. This method is
easy and reliable and has been applied successfully by
many people to more than 50 different cell lines from
A. Principles of the Method
This method is a combination of the two methods
described by Worton and Duff (1979), the suspension
method generally used for nonadherent and adherent
cells and the "in situ
" method that has been specifically
developped for adherent cells (Cox et al.
Peakman et al.
, 1977). When we tried to prepare
metaphases from the well-polarized hybrid cell line
WIF-B (Shanks et al.
, 1994), we only succeeded with the
" one. We performed many assays using the
suspension method and they all failed: from two to
three petri dishes containing 106
cells/dish in exponential
phase, we obtained, at the best, a dozen
metaphases (yield <10-5
). In contrast, with the in situ
method, the metaphase yield was very good and
attained 1.5% of the total cell number (the maximal expected value for cells with a generation time of 2.5
In the suspension method, mitoses are detached
(mechanically or by proteolysis), whereas in the in situ
method, mitoses stay in place. As polarized WIF-B
cells have a highly differentiated plasma membrane,
organized in different domains, we hypothesized that
mitotic cells from this line are very fragile and are lost
during or after detachment (may be during centrifugation).
Therefore, to isolate metaphases from this line,
a new method was developped that avoids cell detachment,
at least during the early steps. The first steps of
this method (metaphase arrest, hypotonic swelling,
and first fixation) are performed in situ
and it is only
after the first fixation that cells are detached.
B. Advantages of the Method
This method presents several advantages. First, it is
applicable to every adherent cell line and does not
depend on the adherent properties of the mitosis, as
all mitoses (floating and adherent ones) are collected.
Second, as with the in situ
method, the yield in
metaphases is very good (Fig. 1A) and therefore the
metaphasic preparations are representative of the
whole cell population. Third, this method requires
fewer cells and is less wasteful in cells than the
suspension method, an advantage for slow-growing
cell lines where mitotic cells are rare or for cells
where the results of karyotyping are required as soon
as possible. Finally, as with the suspension method, the
quality of the metaphase spreading is good (Fig. 1B)
and can be adjusted, whereas with the in situ
which allows only one attempt per culture, the
spreading cannot be controlled and is very sensitive
to cell overcrowding. Moreover, the chromosomes
obtained by this new method are quite suitable for
G banding, Giemsa 11 staining (Buys et al.
(Fig. 1C), and fluorescent in situ
II. MATERIALS AND
|FIGURE 1 Metaphases from HT29 and hybrid WIF lines. (A) HT-29 cells at low magnification;
metaphases were obtained at a high yield as attested by the presence of six metaphases (arrows) among some
30 cells. (B) HT-29 metaphases at a higher magnification to illustrate chromosome spreading. (C) Detection
of human (pale staining, small arrows) and rat (dark staining, long arrows) chromosomes by Giemsa 11 staining
in a metaphase of the human x rat hybrid WIF-B. (D) Detection by FISH (right) of one copy of the human
chromosome 2 (arrow) in a WIF 12-E metaphase that was counterstained with propidium bromide to show
all the chromosomes (left).
Growth medium (available from local suppliers)
Colcemid (10µg/ml; GIBCO, Cat. No. 2465)
Gurr buffer tablets, pH 6.8 (BDH, Cat. No. 33199)
Giemsa solution (Merck, Cat. No. 1.09204.0100)
10-cm tissue culture dishes (Falcon, Cat. No. 3003)
15-ml tubes (Falcon, Cat. No. 352099)
Precleaned, ground edge, microslides (ESCO, Cat. No.
A low-speed centrifuge with a swinging-backet
rotor for 15-ml tubes (as the IEC clinical centrifuge)
is needed for harvesting cells, and a phase-contrast
microscope is needed for examining cells and slides.
III. PROCEDURES A. Cleaning and Frosting of Microscope Slides
B. Optimization of Growth
- Dip microscope slides in a jar containing 100ml
methanol plus two to three drops of concentrated
HCl for 12-24 h.
- Dry the slides one by one, place them in an appropriate
jar, and freeze them at -20°C for at least 4h
and at the most 7 days.
Grow cells in 10-cm Petri dishes in appropriate
medium so that cells will be in midexponential phase
with many mitoses on the day of harvest. Renew the
dishes with 10ml of medium the day before the
C. Metaphase Arrest
Add 0.2ml of colcemid for 1 h at 37°C to the dish
containing the maximal number of mitotic cells. The
other dishes can be used later if necessary.
D. Hypotonic Swelling
- Hypotonic solution (0.075M KCl): add H2O to 0.56g
KCl to make 100 ml
- Fixative: Make fresh 3:1 methanol:glacial acetic
acid; keep it at -20°C in an appropriate closed glass
vessel and transfer to 4°C just before use.
- Collect the culture medium of the Petri dish in a
15-ml tube and centrifuge at room temperature, at
approximatively 1000rpm (200g) for 5min, to collect
the floating mitoses.
- Immediately after starting centrifugation, add
5ml of warm (37°C) hypotonic solution to the cells
in the petri dish.
- Incubate the cells in the hypotonic solution at
- As soon as the centrifuge stops, aspirate and
discard the supernatant and add 1 ml of warm hypotonic
solution to the pellet (depending on the cell line
this pellet could be well visible or almost nonexistent).
Resuspend the pellet rapidly by pipetting and add this
suspension to the cells in the Petri dish, which now
contain all the starting cells. Incubate at 37°C for a
further 20-30 min.
: The swelling of the cells can be verified by examination
under a microscope.
E. Fixation Steps
F. Spreading and Air Drying
- Add very carefully to the petri dish 6ml of cold
(4°C) fixative with a glass pipette and place the dish
on ice for 10 min.
- Detach the cells from the dish with a glass Pasteur pipette by repeated pipetting. Depending on
the cell line, this step could be very easy or more laborious.
Control the detachment under the microscope.
If the cells are very sticky, try gentle use of a cell
- Transfer the cold cell suspension to a cold (4°C)
- Centrifuge at approximatively 1000rpm (200g)
for at least 5 min.
- Remove all but about 0.2ml of supernatant and
resuspend gently by shaking cell pellet in this small
volume (no pipetting). Add 1 ml of cold fixative slowly
and then another 4ml of cold fixative and leave on ice
for at least 5 min.
- Repeat steps 4 and 5 (with a smaller volume of
fixative) twice; the fixative can be added more quickly
on the last fixations. At this point preparations can
be stored overnight at 4°C or slides can be made
Spreading is done by air drying onto frozen slides.
- Centrifuge the fixed cells at approximatively
1000rpm (200g) for at least 5min.
- Remove the supematant and gently resuspend
the pellet in about 0.5 ml of cold fixative (make fresh
fixative if cells have been stored overnight).
- Using a Pasteur pipette, drop two to three drops
of the suspension at the top of a frozen slide inclined
20-30° from vertical. Let the drops run down the
length of the slide as they spread. Wipe excess liquid
from the underside of slide and let it dry at room
- Check this first slide under phase contrast so that
adjustments can be made on subsequent slides if cells
are too crowded or if the spreading is poor.
As the present technique was developed to verify
the cell chromosomal content, we use Giemsa "solid staining" (Worton and Duff, 1979), which gives
uniform staining of the chromosomes and makes it
easy to count them. However, other staining methods
can be applied (Fig. 1).
- Gurr buffer, pH 6.8: Dissolve one Gurr buffer tablet,
pH 6.8, in 1 liter H2O
- 5% Giemsa: Add 5ml of Giemsa to 95ml of Gurr
buffer (or 10mM phosphate pH 6.8)
- Dip the slides in 5% Giemsa at room temperature
for 15 min.
- Rinse twice with water and let dry at room
|FIGURE 2 Chromosomal content of HeLa cells.
were prepared from HeLa cells (ATCC
collection) cultured routinely
in three different laboratories
(A-C). The chromosomal content of
each cell population
is different, and in all cases the chromosome
lower than expected (published mean chromosome
number 82, range 70-86). Note the very heterogeneous
HeLa cells in C. These results show how
cells of a same line can
change and drift.
Using this method, metaphasic preparations from
of a panel of well-known and frequently used lines,
including polarized lines (Caco-2, MDCK, HT-29) and
hepatic ones, were isolated and analyzed (Table I). Some lines (BW1-J, cl l-D, HT-29) displayed a chromosomal
content similar or very near to that published
previously, but the mean number of chromosomes, as
the range, of other lines (Caco-2, HeLa) differed greatly
from those published. Morever, in some cases, big differences
were observed from the same line obtained
from different laboratories. This was the case for HeLa
cells (Fig. 2) and, to a lesser extent, for MDCK. In this
latter case, one population over the three tested was
very heterogeneous and greatly differed from the two
others. Although both L and HeLa lines were established
a long time ago (in 1940 and 1951, respectively)
and thus cultured for a very large number of generations,
the first line seems to be very stable, whereas the
second one has considerable drift.
- Use only glass pipettes for adding the fixative and
for detaching cells after the first fixation. Fluffy
and fuzzy chromosomes are obtained using plastic
- Well-adherent cells can be detached by gentle
pipetting at the end of the hypotonic shock before
- The presence of a thin layer of cytoplasm embedding
the chromosomes can be avoided by performing
additional fixations and by improving the
spreading (increase the distance between the drop
and the slide and adjust the angle of the slide).
I thank C. Delagebeaudeuf for pushing me to
develop this method, C. Hamon-Benais, and V. Bender
for the illustrations, and L. Sperling for careful reading
of the manuscript. This work was supported in part by
the Curie Institute (PIC Signalisation Cellulaire Grant
914), CNRS, and INSERM (contrat Prisme 98-09).
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