|I. General information|
|Background:||This line is a clone of cytotoxic T cells derived from a C57BL/6 mouse.|
|Species:||Mus musculus, mouse|
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|Culture Medium:||RPMI-1640+1mM sodium pyruvate+10%FBS+10% T-STIM with Con A
We strongly suggest to purchase the complete medium from Cobioer.
|Receptor Expression:||interleukin 2 (IL-2), expressed|
|Comments:||The cells are dependent upon IL-2 for growth, and can be used to assay for IL-2. Immediately after thawing (or after overgrowth of a culture) the culture will appear to have few or no viable cells; this is normal.
Depending upon the source and potency of the IL-2, it may take up to three weeks before the cells are ready to subculture. T-STIM with Con A (rat IL-2 culture supplement from Becton Dickinson) may be used or the rat factor may be prepared as described below. The freshly prepared rat factor will usually produce more rapid growth. Tested and found negative for ectromelia virus (mousepox).
T-STIM with Con A (rat IL-2 culture supplement from Becton Dickinson) may be used or the rat factor may be prepared as described on the product sheet. The freshly prepared rat factor will usually produce more rapid growth.
Rat Growth Factor (Laboratory Preparation)
Spleens are removed from female Sprague-Dawley rats weighing 200 g. They are coarsely minced before passing through a No. 60 sieve. Wash cells 2-3 times with RPMI 1640. Resuspend cells at 1-1.5 X 106 viable cells/ml with 100-200 mL/150 cm2 flask in RPMI 1640 containing 1% heat-inactivated fetal bovine serum, 0.05 mM, 2-mercaptoethanol, 15 mM HEPES, 100 units/mL penicillin, 100 mg/mL streptomycin and 1.0 µg/mL concanavalin A.
Incubate at 7°C in a CO2 incubator for 48 hours. Harvest the supernatant by centrifugation at 16,000 x g for 10 minutes at 4°C. Sterilize by filtration using a 0.22 micron membrane.
Store at -60°C. Avoid freeze-thaw cycles. Rat growth factor stored at 4°C up to one month has retained its quality. The growth factor is added to the medium just before use. Expect 1-2 X 108 cells per spleen which yields 100-200 mL of growth factor.
For more information, please contact Cobioer (4008-750-250).