***Preparation of metaphase chromosome spreads from adherent cells or
lymphocytes.***

*Equipment and reagents*

· Mouse Interleukin-2 (Boehringer Mannheim)

· Phytohemgglutinin M-form (Gibco BRL)

· *KaryoMax Colcemid* (Gibco BRL)

· Trypsin--EDTA (Gibco BRL)

· KCl solution (0.056 M or 0.075 M in H~2~O)

· Methanol:Glacial acetic acid at 3:1 freshly made each time

· 75 cm^2^ tissue culture flasks

· Centrifuge conical tubes

· Microscopy slides

· Pasteur pipettes

*Method for adherent cells*

1\. Passage 10^6^ cells/ml to a 75 cm^2^ flask containing 5ml of regular
growth medium 2 to 3 days prior to performing the chromosome spreads.

2\. Feed cells with fresh media 12-14 h prior to harvesting.

3\. Add Colcemid to a final concentration of 0.02 g/ml; incubate at 37 C
for a 3-4 h.

4\. Transfer the medium to a centrifuge tube and rinse the flask with
1.5 ml PBS (without Ca^2+^ and Mg^2+^). Detach the cells with 0.5 ml of
trypsin. Collect the cells and rinse the flask with 1.5 ml PBS 2 times.

5\. Spin down the cells at 160 g for 10 min.

6\. Aspirate the supernatant. Leave about 0.5 ml of supernatant and
resuspend the pellet tapping the bottom of the tube gently with your
finger.

7\. Add 5 ml KCl 0.056 M solution. Pipette up and down to break any
clumps. Leave at room temperature for 30 min.

8\. Spin down the cells at 160 g for 10 min.

9\. Aspirate the supernatant leaving 1 ml of the hypotonic solution.
Resuspend pellet and add 1ml of methanol:glacial acetic acid (3:1)
solution^a^. Spin down the cells at 160 g for 10 min.

10\. Aspirate the supernatant and resuspend the pellet in 3 ml fixative
again. Add slowly and mix the cells as before for the first 1ml.

11\. Spin down the cells at 160 g for 10 min.

12\. Aspirate the supernatant. Repeat steps 10-11 2 more times.

13\. Resuspend pellet in small volume of fixative (typically less than
500ml), until the cell suspension looks slightly milky.

14\. Take up a small quantity of cell suspension in the Pasteur pipette.
Hold the pipette vertically with the end about 10 cm above the slide.
Release a single drop form this height onto the slide. Let air dry^b^.

*Method for suspension cells*

1\. Seed 10^6^ cells/ml in a 75 cm^2^ flask containing 5ml of regular
growth medium supplemented with 100 l of phytohemagglutinin* *and/or 20
U/ml of mouse interleukin-2^c^.

2\. Incubate the cell culture at 37 C for 72 h. Shake the flask gently
1-2 times per day.

3\. Add Colcemid (10 g/ml) about 45 min before harvesting.

4\. Transfer the cells into a centrifuge tube.

5\. Continue as in step 5 of *protocol for adherent cells*, but use KCl
0.075 M solution.

^a^It is very important to disrupt the pellet before addition of the
fixative and to add the fixative only one or two drops at a time tapping
the bottom of tube to mix the cells all the time.

^b^The room temperature and humidity are important factors to be
considered when making the chromosome preparations. Chromosomes will
spread poorly in a worm and dry environment. If necessary breath on the
slide before dropping the suspension. Alternatively, place the dry
slides in the freezer for 30-60 min and drop the cell suspension onto
these cold and wet slides.

^c^Phytohemagglutinin* *an mouse interleukin-2 are mitogenic agents.
Human lymphocytes require only phytohemagglutinin, whereas mouse
lymphocytes grow better with a combination of both of them.