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 Factorxa0 (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 10^6xa0 viable cells/ml with 100-200 mL/150 cm² flask in RPMI 1640 containing 1%xa0 heat-inactivated fetal bovine serum, 0.05 mM,xa0 2-mercaptoethanol, 15 mM HEPES, 100 units/mL penicillin, 100 mg/mL streptomycin and 1.0 µg/mL concanavalin A.xa0xa0xa0xa0xa0xa0xa0xa0xa0

Incubate at 37°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 filtrationxa0 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.

The base medium for this cell line is ATCC-formulated RPMI-1640 Medium, (ATCC^®xa030-2001™). To make the complete growth medium, add the following components to the base medium: additional 2 mM L-glutamine; additional 1mM sodium pyruvate; adjust to a final concentration of 10% fetal bovine serum and 10% T-STIM with Con A. T-STIM is available from Becton Dickinson.

Some Important Considerations in Handling CTLL-2, TIB-214xa0

Frozen Cells: Viability immediately after thawing will be 70-80%. Expect viability to be very poor from day 1 to day 4 after culture initiation. Culture will appear to be completely dead. On the third to fifth day following initiation viable cell clusters will begin to appear in suspension. Usually cells will be ready to subculture on the 7th to the 10th day after the ampule is thawed. However, it may take from 2-3 weeks before vigorous growth is observed. It is best to leave the initial culture undisturbed until cells enter their growth phase.

Overgrowth: In the event cell density becomes too great and viability decreases to where culture appears totally dead, the culture may still be rescued. Inoculate a flask at a density of 1 X 10⁴ viable cells/mL.

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Belani R, Weiner GJ. Expression of both B7-1 and CD28 contributes to the IL-2 responsiveness of CTLL-2 cells. Immunology 87: 271-274, 1996. PubMed: 8698390

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Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.

Caputo JL. Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988

Nucleotide (GenBank) : M27960 Mouse interleukin-4 receptor (secreted form) mRNA, complete cds.

Nucleotide (GenBank) : M27959 Mouse (clone B-2) interleukin-4 receptor (membrane-bound form) mRNA, complete cds.