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