stomach:derived from metastatic pleural effusion; supraclavicular and axillary lymph nodes and Douglas cul-de-sac

55 years adult

Asian

male

Yes, in cheek pouches of anti thymocyte serum treated hamsters

No, in nude mice

Protocol: Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. 1. Remove culture medium with floating cells to a centrifuge tube. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum, which contains trypsin inhibitor. 2.. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 10 minutes). Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal. 3. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting. 4. To remove trypsin-EDTA solution, transfer cell suspension to the centrifuge tube with the medium and cells from step #1 and spin at approximay 125 xg for 5 to10 minutes. 5. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels. 6. Place culture vessels in incubator at 37°C.

Subc*tion Ratio: A subc*tion ratio of 1:2 to 1:8 is recommended

Medium Renewal: Every 2 to 3 days

Freeze medium: Complete growth medium, 95%; DMSO, 5%

Storage temperature: liquid nitrogen vapor phase

Atmosphere: 5% CO2 in air recommended

Temperature: 37°C