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Basic Cytogenetic Techniques: Culturing, Slide Making, and G Banding
|Basic Cytogenetic Techniques:
Culturing, Slide Making, and G Banding
During metaphase of the cell cycle, the condensed
chromosomes align along the metaphase plate prior to
chromatid separation and then move along the spindle
fibres to the poles of the spindle. A spindle inhibitor,
colchicine, can be added during metaphase, which disrupts
the polymerisation of the spindle fibres, arresting
the cell cycle at the stage when the chromosomes
are fully condensed. It is possible to increase the
number of metaphases by synchronising the cell cycle
with a chemical blocking agent such as thymidine. The
addition of excess thymidine results in the inhibition
of DNA synthesis and the cells collecting in S phase.
When the thymidine block is removed by washing the
cells in fresh medium or by the addition of deoxycytidine,
the synchronised cells progress to M phase and,
by careful timing of the addition of colchicine and subsequent
harvest, a large number of longer chromosomes
in early metaphase can be collected.
|FIGURE 1 A metaphase from a synchronized blood
Courtesy of Lyndsey Connell, Oxford
In order to visualise the chromosomes, these cells
are harvested with hypotonic and fixative stages. The
hypotonic solution swells the cells, allowing the chromosomes
to separate. The cells are fixed with glacial
acetic acid/methanol, which removes water and
hardens the cell membrane. The final fixed cell suspension
is dropped onto a microscope slide where
evaporation of the acetic acid/methanol fixative
results in bursting of the cells and spreading of the
metaphase chromosomes. Humidity plays a key role
in the rate of evaporation of fixative and control of
chromosome spreading, hence an optimal microenvironment
is vital to the production of well-spread
Routine G banding with trypsin gives each chromosome
a distinct banded pattern that allows identification
of discrete regions along each chromosome
and the opportunity to screen for chromosomal imbalance
of at least 3-5Mb by standard microscopic
Diagnostic cytogenetic investigations are used routinely
within the clinical setting to screen for chromosomal
imbalance in a wide range of patients. These
methods describe a synchronised suspension culture
of blood and an unsynchronised adhesion culture of
II. MATERIALS AND
RPMI with 20mM
HEPES (52400-017), penicillin
(10,000 U/ml)/streptomycin (10,000 µg/ml)/glutamine
(29.2mg/ml) (10378-016), trypsin 2.5% (15090-046),
phytohaemagglutinin (10576-015), Hanks BSS (14180-
046), and colcemid (15212-012) are from GIBCO. Hams
F10 (N6013), thymidine (T9250), 2-deoxycytidine
(D3897), colchicine (C9754), EDTA 0.02% (E8008),
amphotericin B (A9528), and phosphate-buffered
saline (PBS), pH 7.4 (P3813) are from Sigma. Heparin
(101929) is from ICN. Ultroser G (15950-017) is
from Biosepra. Fetal calf serum is from various
sources, including Sigma, GIBCO, and Seralab.
Methanol (101586B), glacial acetic acid (10001CU), KCl
(101983K), Leishman's stain solution (350226N), buffer
tablets, pH 6.8 (331992P), XAM mountant (361194V),
and washed glass slides (406/0181/02) are from BDH.
Difco trypsin (215320) is from Becton-Dickinson.
Virkon (R330003/Q) is from Radleys. Tissue culture flasks (25cm2
, Nunc 163371), blood culture tubes
(Falcon 35-2027), 1-ml plastic pastettes (Alpha
LW4010), sterile petri dishes (Sterilin 101R20), sterile
scalpels (Swan Morton) and needed. Gas (5%
/95% air) is required if a CO2
incubator is not
Equipment includes a class II laminar flow hood,
incubator, centrifuge, inverted microscope, light microscope
with phase 10 and 100x objectives, hot box oven,
hot plate. Local humidity control can be helped by
humidifiers, dehumidifiers, or controlled environment
A. Synchronised Cell Culture of Human
Blood should be collected into heparinised, ideally
lithium heparin, tubes to prevent coagulation. Although
whole blood is cultured, T lymphocytes are
used routinely for blood cytogenetic investigations. As
these cells are not spontaneously dividing, phytohaemagglutinin
(PHA), a mitogen, must be added to
transform the T lymphocytes and start cell division.
PHA is only required during the first 24h of cell
culture and so is omitted from postsynchronisation
release medium. Blood is routinely cultured for 72h.
Day 1 Establishing Cultures
- Phytohaemagglutinin must be used within 2-3 weeks
- Phosphate-buffered saline pH 7.4: Add one sachet to 1
litre distilled water
- Working stock solutions: Heparin (1250 units/ml in
distilled water), thymidine (10mg/ml in PBSA), 2-deoxycytidine (10µg/ml in distilled water), and colchicine (250µg/ml for blood in distilled water)
- Complete medium: 100ml RPMI, 20ml fetal calf
serum, 1ml penicillin/streptomycin/glutamine,
0.2 ml heparin, and 2 ml phytohaemaglutinin
- Release medium: 100ml RPMI, 10ml fetal calf serum,
2ml penicillin/streptomycin/glutamine, and 2ml
- KCl hypotonic: 0.056 M (4.2 g/litre)
- Fixative: 3:1 methanol:glacial acetic acid
Day 3 Synchronisation.
- Dispense 5 ml of complete medium into a labelled
sterile culture tube. Add 0.25ml of blood with a
sterile plastic pastette. Replace lid and invert to mix.
- Place in a culture rack and incubate in a 37°C incubator.
A culture rack with a slanted edge allows
the tubes to lie at a 20-30° incline and increases
the media/cell pellet surface area for improved
exchange of gas and nutrients.
Add 0.2ml thymidine to
each culture in the late afternoon and reincubate for
17-18 h overnight.
Day 4 Synchronisation Release and Harvest
B. Unsynchronised Cell Culture of
- Centrifuge cultures at 1200rpm for 5min.
Remove the supernatant from the culture tube, leaving
approximately 1cm depth of supernatant. Add 4ml
release medium and resuspend cells. Note time of
release and reincubate.
- Four hours after synchronisation release, add
0.1 ml colchicine per tube, invert culture tubes to mix,
and reincubate cultures.
- After 20min incubation, centrifuge at 1200rpm
for 5 min. Remove supernatant, leaving approximately
0.5 ml in the tube. Resuspend cells and gently add 5 ml
KCl hypotonic. Replace lid and invert the tube to
ensure thorough mixing. Reincubate culture tubes for
- Centrifuge at 1200 rpm for 5 min. Remove supernatant.
Resuspend cells thoroughly. Very gently, add
5ml fixative (drop by drop at first and then more
quickly) whilst continuously mixing the cell pellet
either by flicking the tube or by mixing on a whirlimixer
to prevent cell clumping. This stage is critical in
producing clean preparations.
- Centrifuge culture tubes at 1200rpm for 5min.
Pour off supernatant. Resuspend cells thoroughly and add 5ml fixative. Centrifuge culture tubes again,
resuspend cells, and add 5 ml fixative. If the cell suspension
looks faintly brown, repeat centrifuging and
fixing once more before slide making. The cells are
ready for slide making or may be stored at -20°C.
Tissue viability is improved if the tissue sample is
transported in transport medium supplied by the
Day 1 Establishing Cultures
- Working stock solutions: Heparin (1250 units/ml), amphotericin B (0.25mg/ml), and colchicine (500µg/ml for tissues)
- Transport medium: 100ml Hams F10, 1ml penicillin/
streptomycin/glutamine, 0.2 ml heparin, and
1ml amphotericin B
- Complete medium: 100 ml Hams F10, 20 ml fetal calf
serum, 1ml penicillin/streptomycin/glutamine,
1ml amphotericin B, and 2ml Ultroser
4. Trypsin/EDTA: 10ml of trypsin 2.5% added to 100ml
- KCl hypotonic: 0.056 M (4.2 g/litre distilled water)
- Fixative: 3:1 methanol:glacial acetic acid
Days 6-8+ Processing and Harvesting of Cultures
- Remove the tissue from the transport medium
with a sterile pair of forceps and place in a sterile
plastic petri dish. Using a sterile plastic pastette, add
a few drops of medium to the tissue to prevent the
sample from drying out. Mince the tissue finely with
a sterile scalpel into ~0.5- to 1-mm3 pieces.
- Using a pastette, resuspend the minced tissue in
a small volume (~0.25 ml) of medium and place on the
growth surface of two labelled 25-cm2 flasks. Ensure
that the tissue pieces are as evenly spread as possible.
Gently invert the flask so that tissue adheres to the
surface in a minimal amount of medium. Add 5 ml of
complete tissue medium to each flask by running the
medium down the lower side of the inverted flasks.
Gas (5% carbon dioxide/95% air) the flasks if using a
standard non-CO2 incubator, replace lids, and incubate
- After several hours (or overnight if cultures are
set up late in the day), carefully turn the flasks the right
way up so medium now covers the tissue.
- Cultures are examined for the first time when
they are 6-8 days old. Replace the medium with
5ml of complete tissue medium. Gas (5% carbon
dioxide/95% air) the flasks if using a standard non-
CO2 incubator, replace lids, and incubate at 37°C.
- When sufficient cells have grown from the
explants (covering one-half to two-thirds of the flask),
the cells can be directly harvested or subcultured. Subcultured
cells usually give a higher mitotic index.
Harvesting of Cultures.
- Prewarm trypsin/EDTA to 37°C. Remove culture
medium and gently rinse flask with ~1ml of
trypsin/EDTA. Add 0.5ml of prewarmed trypsin/
EDTA and reincubate for several minutes. Examine
the flask under low magnification on an inverted
microscope. The cells should appear either rounded up
or floating in the medium. Tap the flask lightly two or
three times to dislodge any remaining cells. Add 1ml
of medium and divide the cell suspension between
two new labelled flasks.
- Add 5ml of complete medium to each flask,
including the original, which can be retained as a
backup. Gas (5% carbon dioxide/95% air) the flasks if
using a standard non-CO2 incubator, replace lids, and
incubate at 37°C.
Subcultured flasks are
usually fed with 5 ml fresh medium the following day
and harvested on the second day. The cells should
be in logarithmic growth phase with a high mitotic
C. Slide Making
- Add 0.1ml colchicine to each flask and reincubate
for 2h. Prewarm trypsin/EDTA to 37°C.
- After 2h, examine the cells under low magnification
with an inverted microscope. A number of
rounded, dividing cells should be present. Pour off the
medium from the flask into a labelled centrifuge tube.
Add 1ml of trypsin/EDTA and rinse the flask, adding
this to the centrifuge tube. Add 0.5-1ml of trypsin/
EDTA to each flask and incubate for 2-5min. Tap
the flask two or three times. Examine the flasks to
check that cells are in suspension. Add 2 ml of culture
medium to the flask and transfer all medium and cells
to the centrifuge tube.
- Centrifuge the tube at 1200rpm for 7min. Pour
off the supernatant from the tube. Resuspend the pellet
by flicking the tube. Add 5 ml KCl hypotonic solution
to the tube and invert gently to mix. Centrifuge at
1200rpm for 7 min. Pour off the hypotonic, resuspend
the cell pellet, and gently and slowly add fixative
whilst flicking the tube to prevent cell clumping. After
approximately 0.5ml has been added, top up more
quickly to 5 ml. The cells are ready for slide making or
may be stored at -20°C.
It is advisable to wipe the precleaned slide with a
tissue soaked in either methanol or fixative immediately
prior to slide making. Optimal conditions are
40-45% humidity in temperatures of 20-22°C.
Fixative solution: 3:1 methanol: glacial acetic acid
D. G Banding
- Centrifuge cell suspensions at 1200rpm for
5min. Pour off supernatant and resuspend the cell
pellet by tapping the tube and add three drops of
- Using a plastic pipette, place a single drop of cell
suspension in the middle of a labelled slide from a
height of I cm. The slide can be held fiat, allowing the
drop to spread as a circle or the slide can be held at
20-30° so that the drop runs down the length of the
- Add a single drop of fixative from the same
height in the same manner when the area of cells
becomes visible as a cloudy area and Newton's rings
appear on the periphery. Allow the fixative to
- Check the cell density and metaphase spreading
using a phase microscope.
- If the slide has sufficient metaphases of an appropriate
length, continue with slide making. If the cell
density is too high, add a few more drops of fixative
and check by making another slide. If the cell density
is too low, add 5 ml fixative, centrifuge the suspension
again, and repeat but add less fixative. Cell spreading
can be altered by changing cell density, local humidity,
or the rate of drying. Underspreading can be improved
by breathing on the slide before adding the cell suspension,
adding two drops of fixative to the cells on
the slide, or placing the slide on a wet paper towel.
Overspreading can be reduced by omitting the second
drop of fixative, using cold fixative cooled in a freezer
before use, or increasing local air flow and speed of
- Add 5 ml fixative to any remaining cell suspension
and store at -20°C.
- Age slides either overnight by putting in metal
racks in a hot box oven at 70°C or for 1h on a hot plate
at 80°C or on the bench for several days.
- Hanks pretreatment solution: 8ml of Hanks BSS (10x)
plus 40ml of distilled water
- Phosphate-buffered saline pH 7.4: Add one sachet of
powder to 1 litre of distilled water
- Banding trypsin solution: Rehydrate bottle of Difco
trypsin with 10ml of distilled autotoclaved water.
Add 1ml of trypsin solution to 40ml PBSA.
- pH 6.8 buffer solution: Add 1 tablet to 1 litre of distilled
- Leishman's staining solution: 40ml, pH 6.8, buffer
solution plus 8 ml Leishmans' stain
- Place a test slide in the Hanks pretreatment solution
for 3 min. Rinse the test slide in tap water for 5 s.
Place the slide in the trypsin solution for 33s (move
slide gently in the solution). Whilst slide is in the
trypsin solution, remove surface film from the stain by
skimming with a piece of filter paper. Rinse slide in tap
water. Place the slide in Leishman's staining solution
for 2min. Rinse slide in tap water. Blot carefully with
filter paper and leave to dry or dry on a hot plate for
a short time.
- When dry, place three drops of XAM or similar
mountant onto the slide along its length. Place a 22 x
50-mm coverslip onto the slide and blot gently to
remove excess mountant.
- Assess banding using the light microscope at
1000x magnification and adjust times for Hanks,
trypsin, and stain as appropriate. If the chromosomes
appear generally grey with indistinct banding,
increase the Hanks pretreatment time. If the chromosomes
are bloated and the chromatids appear to be
separating, reduce trypsin time. If they appear generally
dark with little banding, increase the trypsin time.
Staining should be modified so there is a clear distinction
between pale staining and dark staining regions
of the chromosomes.
- All culture work should be performed under the
appropriate containment level (e.g., class 2 laminar
- All waste material from cell cultures should be
decontaminated prior to disposal. Waste media and
hypotonic can be decontaminated in 2% Virkon for a
minimum o f 10 min.
- Fetal calf serum is variable in supporting good
cell growth and should be batch tested prior to bulk
purchase and use. It is advisable to combine smaller
aliquots from two different batches or suppliers rather
than a single volume from one supplier in preparing
- All complete culture media should be stored at
4°C and ideally used within 1 week of preparation.
- Our laboratory routinely uses colchicine. Many
laboratories use the less toxic colcemid, adding 0.1 ml
of stock solution to 5 ml culture.
- The methanol/glacial acetic acid fixative should
be freshly made before harvesting and slide making.
- Slides should be grease free to ensure optimal
chromosome spreading. Precleaned slides may be
If the first fixation stage of a blood culture results in
a gelatinous cell suspension, add an equal volume of
distilled water, mix, recentrifuge, and continue with