**TITLE: TRYPSIN-GIEMSA STAINING (G-BANDING)**

**SPECIMEN**: Slides prepared from fixed cell suspensions from harvested
cultures of bone marrow, spleen, lymph node, tumor cell lines, or
primary cultures of tumors.

**REAGENTS**:

1\. [Isoton Solution]{.underline}:

Isotonic Saline Diluent (VWR #73000-086). Adjust pH to 6.8.

2\. [Trypsin Solution]{.underline}:

Trypsin powder, Difco 1:250 (VWR #DF0152-13-1) 100 mg

Isoton, pH adjusted to 6.8 100 ml

Stir on a magnetic stirrer for 1-2 hours. Allow the solution to cool to
RT before banding. Unused solution may be stored at 4^o^C for 2-3 days,
and warmed to RT prior to use.

3\. Fetal Bovine Serum.

4\. [Gurr Buffer Solution]{.underline}:

#  Gurr Buffer tablets, pH 6.8 (Bio-Medical Specialties #33199-2P ) 1 tablet

Double Distilled H~2~0 1L

Stir on a magnetic stirrer until tablet has dissolved. Adjust pH to 6.8.
Solution may be stored at room temperature for up to 6 months.

5\. [Giemsa Stain Solution]{.underline}:

#  Gurr\'s Improved R66 Giemsa Stain (Bio-Medical Specialties #35086 2M) 1.5 ml

#  Gurr Buffer Solution, pH adjusted to 6.8 50 ml

Mix both reagents well in a Coplin jar. The thin layer of
film/undissolved material that rises to the surface may be skimmed off
with a Kimwipe, or a clean microscope slide. Working solutions of stain
should be made daily.

**PROCEDURE**:

1\. Age air-dried slides for 1-3 days at room temperature or in the
drying oven (50-60^º^C), then place on slide warmer (55-60^º^C) for 2-4
hours. Age fresh slides by placing on a 55-60^º^C slide warmer for 4
hours prior to staining. Allow slide to cool to room temperature or
until slightly warm and start banding procedure immediately. Do not
allow slides to sit at room temperature after removal from the slide
warmer.

2\. Just prior to banding, prepare a series of Coplin jars containing
the following solutions:

Jar 1. Trypsin solution (50 mls).

Jar 2. 45 mls Isoton (pH 6.8) with 5 mls fetal bovine serum (serum will
inactivate trypsin).

Jar 3. 50 ml Isoton.

Jar 4. 1.5 ml Giemsa stain in 50 mls Gurr buffer (pH 6.8).

Jar 5. 50 mls Gurr buffer (pH 6.8).

3\. Immerse slide in the trypsin solution for 10-25 seconds. The slide
should be agitated continuously by lifting the slide out of the solution
and replacing it carefully. [The trypsin time may vary from sample to
sample; therefore, one slide from each culture should be tested to
determine the optimal exposure time at the start of
staining.]{.underline} A single slide may be used to test two trypsin
times by immersing the bottom half 2-10 seconds longer than the top
portion of slide. The test slides are examined under the microscope
after staining to select the optimal exposure time.

4\. Quickly dip the slide 5-7 times in jar 2 containing Isoton and FBS.

5\. Dip slide 5-7 times in Isoton (3rd jar).

6\. Stain in buffered Giemsa solution for 8 minutes. The test slides are
examined under the microscope after staining to select the optimal
staining period. Increase or decrease time as needed.

7\. Drain slide and rinse by dipping in Gurr buffer (jar 5).

8\. Blot back of slide dry, invert slide by placing the frosted end
down, and allow slide to dry on slide rack in a vertical position.
[Do]{.underline} [not]{.underline} blot front of slide.

9\. Change solutions after 20 slides are processed. Slides should be
monitored as they are prepared, since it may be necessary to vary the
length of trypsin treatment or staining.

**PROCEDURE NOTES**:

If the cells are [under-trypsinized]{.underline}, the chromosomes will
have:

\(1\) Poor differentiation of bands with uniform, darkly-stained
chromatids.

If the cells are [over-trypsinized]{.underline}, the chromosomes will
appear:

\(1\) Fuzzy

\(2\) Chewed or frayed

\(3\) Bloated

Variability in metaphase spreading and length of chromosomes will
influence the optimal trypsin exposure period. The trypsin time may vary
for metaphase cells from different tissues. Also, temperature and pH of
the trypsin solution can affect trypsin time; if the room temperature is
warmer than usual (25-27^o^C), a shorter trypsin time may be necessary
(the activity of the enzyme increases with higher temperatures and
decreases with lower temperatures). Trypsin is most active at a slightly
basic pH (pH 8.0). These and other variables can result in over- or
under-trypsinization. Therefore, at least one slide/culture should be
assessed by examining 3-10 cells with different spreading and chromosome
length. It is also important to examine cells from different parts of
the slide, as chromosome spreading and banding may differ along the
length of the slide.

**Submitted by:**

Michelle M. Le Beau, Ph.D.

University of Chicago

mlebeau@medicine.bsd.uchicago.edu