Preparation of tumor cells

Grow cells in complete medium and exclude any contamination When cells are 70-80% confluent, 3-4 hrs before harvesting, replace medium with fresh medium to remove dead and detached cells. 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. Count cells using a hemocytometer. Using trypan blue staining to exclude dead cells. 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. 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

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

Preparation of the injection

Clean and sterilize the inoculation area of the mice with ethanol and/or iodine solutions Use 1-cc syringe and a 27- or 30-gauge needle 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. Inject cells (3.0 x 106 ) subcutaneously (s.c.) into the lower flank of the mice. Therapy can be started after 1-3 weeks when the tumors have reached an average volume of ~50�60 mm3 . Tumor diameters are measured with digital calipers, and the tumor volume in mm3 is calculated by the formula:

Volume = (width)2 x length/2