Chromosome, Microdissection and Microcloning
23
A few drops of heparinized blood are
mixed with 5 mL of prewarmed chro-
mosome 4A medium or growth medium
containing 0.2 mL of phytohemagglutinin
solution in sterile polypropylene tubes.
Cultures are set up in duplicate and placed
in an incubator at 37
C for 72 h. A
stock solution of colcemid (diacetylmethyl-
colchicine) is added to each tube to a
Fnal concentration of 0.05
µ
gmL
1
and
incubated for an additional 30 min. The
cell pellet is prepared by centrifugation at
800
g
for 10 min and resuspended in warm
(37
C) 5 mL hypotonic solution (0.075 M
KCl) and incubated at 37
C for 10 min.
The cell pellet is prepared again by cen-
trifugation at 800
g
and is resuspended in
5 mL of cold 3 : 1 methanol–acetic acid
Fxative on ice. Cells are dropped onto
large, (35 mm
×
50 mm) cold, wet cover-
slips using a Pasteur pipette. Coverslips
are examined Frst on a phase microscope
to determine whether the spreads are at
the correct density. The coverslips are kept
at
70
C in ethanol for a few hours or un-
til use. Immediately before use, coverslips
are immersed in ice-cold distilled water. If
monolayer tissue culture cells are used to
prepare chromosomes, the cells are seeded
at low density onto plastic petri dishes for
24 h; then the medium is removed and
replaced with fresh medium. Colcemid so-
lution is added 48 to 72 h later and cultures
are incubated for 10 min to 1 h at 37
Cina
5% CO
2
incubator. Cells are removed from
the culture plate or flask and processed as
described for lymphocytes.
Among the numerous effective mitotic
spindle inhibitors, colcemid is particularly
useful, as it arrests cells at the M/G2
interphase. Since chromosomes become
more condensed and shorter at the end
of metaphase, short incubation times with
colcemid (10–60 min) tend to yield more
extended chromosomes. This is preferable,
particularly when one is dissecting small
chromosomes or attempting to dissect mi-
nor bands. The use of agents that promote
chromosome extension and enhance DNA
nicking (e.g. ethidium bromide) should
be avoided.
2.1.2
Hypotonic Solution
The hypotonic solution swells the nucleus
and cytoplasm, breaks intrachromosomal
connections, and allows a better separa-
tion of chromosomes when the cells are
smashed on a coverslip. Hypotonic treat-
ment of cells results in degradation of DNA
from chromosome spreads. Therefore, the
shortest length of exposure to hypotonic
solution is preferable. Lymphocyte cul-
tures usually can be treated for 10 min
at 37
C to produce adequate spreads,
whereas monolayer cell lines are usually
treated for 30 min at 37
C. Prolonged hy-
potonic treatment results in cell lysis and
dispersion of chromosomes over a large
area. Less than optimum hypotonic treat-
ment results in tightly packed metaphase
spreads that may not be suitable for mi-
crodissection.
2.1.3
Chromosome Fixation
Chromosome Fxation serves to arrest
chromosomes at a speciFc stage in the
cell cycle with little distortion of mor-
phological detail. Optimum Fxation also
rids the spreads of cytoplasmic debris.
The most commonly used Fxative is a
mixture of methanol and acetic acid, at
a ratio of 3 : 1 or higher. Acetic acid,
which has excellent penetrability, will
precipitate nucleoprotein, extract consid-
erable amounts of chromosomal proteins,
and remove cytoplasmic constituents from
metaphase spreads. Mixtures of acetic acid
and methanol produce minimal swelling
or condensation of chromosomes. Acetic
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