**TITLE**: **PREPARATION OF SLIDES OF METAPHASE CELLS FOR CYTOGENETIC
ANALYSIS.**

**SPECIMEN**: Fixed cell suspensions from harvested cultures of bone
marrow, spleen, lymph nodes, tumor cell lines, or primary cultures of
solid tumors.

**REAGENTS**:

1\. Fixative: 3:1, absolute methanol: glacial acetic acid.

Methyl Alcohol Anhydrous AR, Low Acetone, ACS grade, Mallinckrodt,

VWR \# MK302410 **FLAMMABLE** 3 parts

Acetic Acid, Glacial, Baker Analyzed Reagent,

VWR #JT9507-4 **CORROSIVE** 1 part

CAUTION: Methanol is flammable; acetic acid is corrosive and
combustible. Use caution when handling. Fixative is caustic to skin and
mucous membranes. Wash off any spills immediately, flush with water. Use
eye protection. See MSDS.

Mix fixative in a glass stoppered flask or bottle immediately before
initial use. Store at room temperature. Mix by swirling the flask
immediately before each use. Fixative should be prepared fresh for each
harvest, and stored for a maximum of two hours.

2\. 95% ethyl alcohol.

**PROCEDURE**:

Procedures for preparing slides from cell suspensions are numerous, and
vary from laboratory to laboratory. The following technique was
developed by testing multiple variable and methods using murine
hematopoietic cells; however, it may be necessary to modify this
technique to obtain optimal results in other laboratories.

1\. After harvesting, cells can be stored at -20^o^C indefinitely, or
slides can be prepared immediately. In either case, the cells must be
washed in fixative 3-6 times. The number of washes depends upon how
clean the pellet is, and how clear the fixative is after the third wash.

2\. Centrifuge tubes at approximately 1000 rpms for 8 minutes to pellet
the cells. The supernatant is decanted, and the cell pellet is gently
resuspended by tapping the tube. Add 10 mls of fixative to the tube, and
mix the cell suspension by bubbling air through a Pasteur pipette. This
washing process promotes improved chromosome spreading and morphology,
and removes cellular debris.

3\. After the last wash, centrifuge sample and discard the supernatant.
Resuspend cell pellet in enough fixative to produce a slightly milky
cell suspension (the volume of fresh fixative required is approximately
equal to ten times the volume of the cell pellet). Care should be taken
to dilute the sample correctly, since underdilution can result in an
overly dense slide and poor spreading of metaphase cells.

4\. Clean glass microscope slides by immersing in 95% ethanol followed
by wiping with a Kimwipe or other lint-free cloth.

5\. Place one drop of fixative on the top of the slide (below frosted
edge); the fixative will spread and cover the surface of the slide. Care
should be taken not to place more than [one]{.underline} drop of
fixative onto the dry slide, as too much fixative may cause cell nuclei
and metaphase cells to accumulate along the edges of the slide.

6\. Hold the slide at a 60^o^ angle over a steam bath, and drop 4-6
drops of the cell suspension from a Pasteur pipette held 18-24 inches
above the slide, beginning at the top of the slide and moving downward.
Immediately place the slide over a steam bath created by running hot tap
water (45-50^o^C) into a pan. Two glass rods, placed 1 1/2 inches apart
across the rim of the pan, can be used to hold the slides above the
water level in the pan.

7\. The slides are removed from the pan when the fixative has dried from
the upper surface (2-3 minutes). Slides should be aged at room
temperature or in a drying oven (55-60ºC) for 1-3 days prior to
staining. Slides for fluorescence in situ hybridization or spectral
karyotyping analysis should be stored in a sealed box with a desiccant
(Drierite) at 4ºC or --20ºC.

**PROCEDURE NOTES:**

1\. If the metaphase cells are [underspread]{.underline}, one or more of
the following steps may be applied:

• Perform 2-5 additional washes in fixative,

• Increase the angle at which the slide is held while dropping the
suspension,

• Increase the distance between the slide and the pipette,

• Dilute the cell suspension by adding fixative.

2\. If the metaphase cells are [overspread]{.underline}, one or more of
the following steps may be applied:

• Decrease the angle at which the slide is held while dropping the
suspension,

• Decrease the distance between the slide and the pipette.

**Submitted by:**

Michelle M. Le Beau, Ph.D.

University of Chicago

mlebeau@medicine.bsd.uchicago.edu