**TITLE:** **SHORT-TERM CULTURE OF MURINE HEMATOPOIETIC CELLS FOR
CYTOGENETIC ANALYSIS**

**SPECIMEN:** Bone marrow, spleen, lymph node biopsy.

**REAGENTS:**

All reagents are purchased from Invitrogen Life Technologies, except
where indicated.

1\. Culture Medium

[Standard Culture Medium]{.underline}:

70% RPMI 1640,

10% HI-FBS (Heat-inactivated fetal bovine serum),

10% IL-3CM,

10% IL-6CM,

10 mM HEPES (1 ml per 100 mls),

100 units/ml penicillin/100 mcg/ml Streptomycin (1 ml per 100 mls),

37^o^C, pH 7.2-7.3.

Prepared media may be stored for up to 7 days at 4^o^C, and used within
this time period.

IL-3 conditioned media is prepared from the Xg63Ag8-mIL-3 cell line.1

IL-6 conditioned media is prepared from the Sp2/mIL-6 cell line (ATCC).

Cells are cultured in 90% RPMI 1640, 10% FBS, 100 units/ml
penicillin/100 mcg/ml Streptomycin (37^o^C, pH 7.2-7.3) by plating in
75cm2 flasks at a density of 1 X 105 cells/ml. Incubate for 3-5 days
until media is yellow. Remove the media and centrifuge. Filter media
using a 0.2 micron filter,.and store at --20ºC.

[ALTERNATIVE MEDIAS:]{.underline}

[ASM Medium:]{.underline}

500 ml DMEM-High glucose, with glutamine, without sodium pyruvate,

5 ml MEM Non-essential amino acids (10 mM, 100x),

5 ml Na Pyruvate (100x),

10% FBS-Qualified

1.2 ml Bovine Insulin (4 mg/ml),

110 mcl 2-mercaptoethanol (55 mM, final concentration 10^-5^M),

2.5 ml Oxaloacetic acid (SIGMA #07753)

50 ml Medium NCTC-109 10%,

10 mM HEPES (1 ml per 100 mls),

100 units/ml penicillin/100 mcg/ml Streptomycin (1 ml per 100 mls),

37^o^C, pH 7.2-7.3.

Prepared media may be stored for up to 7 days at 4^o^C, and used within
this time period.

[Myelocult medium:]{.underline}

Myelocult (Stem Cell Technologies, M5300). This is a complete medium.

Optional supplements: 5% IL-3CM, 5% IL-6CM, G-CSF (5 ng/ml).

3\. [Dulbecco\'s Phosphate Buffer Solution]{.underline} (Invitrogen Life
Technologies #14040).

4\. [Trypan Blue Stain]{.underline} (Invitrogen Life Technologies
#15250-061)

**PROCEDURE:**

1\. Enumerate cells in the sample using a Hemavet, or by manual counting
with a hemacytometer.

[Hemacytometer Counting:]{.underline}

In a sterile 15ml tube, mix 0.1 ml of trypan blue stain, 0.7 ml of PBS,
and 0.2 ml of sample. Mix well with a Pasteur pipette and transfer a
drop of the suspension to each chamber of a hemacytometer. Count the
total number of cells, and the total number of clear cells without blue
staining in 5 large squares (four corners and center square) in each
chamber, for a total of 10 squares. To determine the percent viable
cells, divide the total number of cells without blue staining by the
total number of cells. To determine the total number of viable cells,
multiply the total number of cells without blue staining by 0.005 to
obtain the number of cells x 10^6^/ml of sample.

2\. Short-term cultures are initiated by incubating ten million cells in
10 ml of standard culture medium (1 x 10^6^ cells/ml) at 37^o^C (5%
CO~2~/95% air, humidified atmosphere) for 24-48 hours. Incubate culture
flasks upright with caps slightly loose. If there are sufficient cell
numbers, initiated 2-4 cultures. If two cultures are initiated, they
should be harvested after 24 hours incubation, and processed using
routine harvesting procedures (see Metaphase Cell Preparation protocol.
Additional flasks can be harvested after 48 hours incubation.

**REFERENCES:**

1\. Karasuyama H, Melchers F: Establishment of mouse cell lines which
constitutively secrete large quantities of interleukin 2, 3, 4, or 5,
using modified cDNA expression vectors. Eur. J. Immunol. 18:97-104,
1988.

**Submitted by:**

Michelle M. Le Beau, Ph.D.

University of Chicago

mlebeau@medicine.bsd.uchicago.edu