CTLL-2 (ATCC® TIB-214)

Organism: Mus musculus, mouse  /  Cell Type: lymphocyte cytotoxic T lymphocyte;  / 

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Organism Mus musculus, mouse
Cell Type lymphocyte cytotoxic T lymphocyte;
Product Format frozen
Morphology lymphoblast
Culture Properties suspension
Biosafety Level 1
Strain C57BL/6
Applications
The cells are dependent upon IL-2 for growth, and can be used to assay for IL-2.
Storage Conditions liquid nitrogen vapor phase
Images
Derivation
This line is a clone of cytotoxic T cells derived from a C57BL/6 mouse.
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 10viable 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 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 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.


Complete Growth Medium The base medium for this cell line is ATCC-formulated RPMI-1640 Medium, Catalog No. 30-2001. 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.
Subculturing
Subculture actively growing suspension cultures before they have reached 2 X 105 cells/ml or the IL-2 will rapidly deplete and the cells will quickly lose viability Use inoculation densities of 1 to 2 X 104 viable cells/mL. Corning® T-75 flasks (catalog #431464) are recommended for subculturing this product.
Medium Renewal: Twice per week

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

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.

Cryopreservation
Freeze medium: Complete growth medium supplemented with an additional 10% fetal bovine serum and 7.5% DMSO
Storage temperature: liquid nitrogen vapor phase
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Name of Depositor S Gillis
References

Gillis S, Smith KA. Long term culture of tumour-specific cytotoxic T cells. Nature 268: 154-156, 1977. PubMed: 145543

Hu SX, et al. Development of an adenovirus vector with tetracycline-regulatable human tumor necrosis factor alpha gene expression. Cancer Res. 57: 3339-3343, 1997. PubMed: 9269991

Mazzaccaro RJ, et al. Major histocompatibility class I presentation of soluble antigen facilitated by Mycobacterium tuberculosis infection. Proc. Natl. Acad. Sci. USA 93: 11786-11791, 1996. PubMed: 8876215

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

Hay RJ, Caputo JL, Macy, ML, Eds. (1992) ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

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

Cross References

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.

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
Other Documentation
FAQ's
  1. TIB-214 morphology and culture condition


    Date Updated: 2/21/2014

References

Gillis S, Smith KA. Long term culture of tumour-specific cytotoxic T cells. Nature 268: 154-156, 1977. PubMed: 145543

Hu SX, et al. Development of an adenovirus vector with tetracycline-regulatable human tumor necrosis factor alpha gene expression. Cancer Res. 57: 3339-3343, 1997. PubMed: 9269991

Mazzaccaro RJ, et al. Major histocompatibility class I presentation of soluble antigen facilitated by Mycobacterium tuberculosis infection. Proc. Natl. Acad. Sci. USA 93: 11786-11791, 1996. PubMed: 8876215

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

Hay RJ, Caputo JL, Macy, ML, Eds. (1992) ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

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