155- Quench trypsin by adding at least 3 volumes of 10% fetal
bovine serum (FBS) containing medium (see Note 21). - Pellet cells by centrifugation for 5 min at 200 × g. Aspirate
medium, wash cells with serum—medium, mix well with
pipette, and save 50 μl aliquot of cells for counting. Pellet cells
by centrifugation for 5 min at 200 × g for final resuspension in
serum free medium according to cell count for mice
injections. - Count the viable cell number by trypan blue viability test using
hemocytometer before the final suspension in medium. Add
50 μl of trypan blue to 50 μl of saved aliquots of cells, mix well,
and count cells using the hemocytometer. - Centrifuge cells at 200 × g for 5 min to pellet cells. Then remove
the medium and resuspend the cells in the medium without
serum at concentrations of 10 × 105 per 200 μl of medium for
subcutaneous xenograft model and 10 × 105 to 10 × 106 cells
per ml of medium for intraperitoneal xenograft model. Keep the
suspended cells on ice before injection into mice (see Note 22). - Do all mouse experiments using protocols in accordance with
the standards and guidelines of the Institutional Animal Care
and Use Committee (IACUC) of the relevant institutions and
national regulations. Maintain all mice under pathogen free
barrier conditions (see Note 23). - Agitate the cell suspension in 50 ml Falcon tube to prevent the
cells from settling and draw up into the syringe (1 ml tubercu-
lin syringe) and needle (25 G × (^1) 1/2), the amount of cells in
serum free medium (200 μl, 10 × 105 per 200 μl of medium)
to be administered (see Note 24).
Hair removal from the flanks of SCID mice should be done
with electrical clipper before subcutaneous injection of cells
(see Note 25). You need two persons to perform the subcuta-
neous injection properly. Gently remove the animal from the
cage and restrain appropriately with holding the skin over the
neck with one hand and the leg with another hand. Identify
anatomical landmarks in order to inject cells into the appropri-
ate area of the flank.
Insert the long needle (25 G × (^1) 1/2) into the subcutaneous
space of the flank and slowly inject the cells creating a single
bubble of cells beneath the skin (see Note 26).
Randomly group mice (usually n = 5 per group) when the mice
have measurable tumor size, usually 2 weeks after injection of
cells.
Treat mice intraperitoneally with vehicle, paclitaxel (20 mg/
kg, two times a week for 2 weeks) [ 23 ] or carboplatin (50 mg/
kg, two times a week for 2 weeks) [ 24 ].
3.2 Preparation
of Mice
and Subcutaneous
Injection of Cancer
Cells
3.3 Subcutaneous
Tumor Growth Assay
Xenograft Models of Esophageal Adenocarcinoma