Abstract: Protocol for estabolishing xenograft tumor models in nude mice or SCID mice. Procedure includes preparation of cells and mice, and injection.

Preparation of tumor cells

1. Grow cells in complete medium and exclude any contamination
2. When cells are 70-80% confluent, 3-4 hrs before harvesting, replace medium with fresh medium to remove dead and detached cells.
3. Remove medium and wash cells with PBS. Add a minimum amount of trypsin-EDTA. Disperse cells and add complete medium (10:1 to 5:1). Centrifuge immediately at or below 1500 rpm for 2-5 min and wash twice with PBS and store cells on cell.
4. Count cells using a hemocytometer. Using trypan blue staining to exclude dead cells.
5. Mix cells 1:1 with trypan blue solution (Trypan Blue: dilute at 0.8 mM in PBS. Store at room temperature. Stable for 1 month.). Viable cells exclude trypan blue, while dead cells stain blue due to trypan blue uptake.
6. Cells should be suspended in a volume so that 300 µl contains required number of cells per injection. Usually, 3.0 x 10⁶ cells are needed per injection.

Preparation of mice

1. Mice should be 4-6 weeks old.
2. Allow 3-5 days acclimatization period after mice have arrived.

Preparation of the injection

1. Clean and sterilize the inoculation area of the mice with ethanol and/or iodine solutions
2. Use 1-cc syringe and a 27- or 30-gauge needle
3. Mix cells and draw the cells into a syringe without a needle. Using a needle causes a strong, negative pressure which can cause cell damage and lysis.
4. Inject cells (3.0 x 10⁶) subcutaneously (s.c.) into the lower flank of the mice.
5. Therapy can be started after 1-3 weeks when the tumors have reached an average volume of ~50–60 mm³.
6. Tumor diameters are measured with digital calipers, and the tumor volume in mm³ is calculated by the formula:

   Volume = (width)² x length/2

Reference
Jacob et al, Gene Ther Mol Biol 2004;8:213-219.