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Fig. 3 Focus formation by Ad5 E1A and E1B . Primary human amniotic fl uid cells
were transduced with ( a ) empty LeGO vector particles, or ( b ) co-transduced with
Ad5 E1A and E1B harboring LeGO vector particles with an MOI of 0.3 particles
per cell. Transduced cells were stained with crystal violet 27 days after transfec-
tion and scanned
1. Grow cells on 10- or 15-cm tissue culture dishes. At the day of
injection, dishes should contain 50-80 % confl uent monolay-
ers. Each transformed cell line should be tested in at least fi ve
mice, and for each animal 1 × 10 6 cells are needed.
2. Wash monolayers two times with serum-free DMEM and
scrape cells into serum-free medium using a sterile plastic cell
lifter. Carefully transfer cell suspension to a conical tube and
centrifuge for 5 min at 800 × g .
3. Remove supernatant and add serum-free medium to make up
a suspension that contains 10 7 cells/mL (1 × 10 6 cells per
injection).
4. Inject 100
3.6.1 Tumor Incidence
and Tumor Growth
in Athymic Mice
L of cell suspension into nude mice subcutane-
ously using a syringe equipped with a 26-G needle.
5. Examine the site of injection weekly for development of tumor
nodules to determine the latency period. After development of
visible (palpable) tumors, measure the length and width of the
tumors and calculate the approximate tumor area. Results are
displayed as tumor incidence (no. of mice with tumors/total
no. of animals) and tumor size (mean tumor area ± standard
errors [mm 2 ]).
μ
For histological analysis, tumors as well as surrounding connective
tissue and adhering skin regions are fi xed in Bouin's solution and
prepared for routine paraffi n histology. Sections (5
3.6.2 Tumor Histology
and Metastasis Assay
m) are stained
with hematoxylin-eosin according to the Masson and Golder
method modifi ed by Jerusalem. Athymic mice injected with trans-
formed cells that induce tumors can also be examined for
μ
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