CTLL-2

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 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.

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C.  Storage at -70°C will result in loss of viability.

1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. It is recommended that the cryoprotective agent be removed immediately.  Centrifuge the cell suspension at approximately 125 x g for 5 to 10 minutes.  Discard the supernatant and resuspend the cell pellet in an appropriate amount of fresh growth medium. Adjust the cell density of the suspension to  1 X 10⁵ viable cells/mL. 
4. Transfer cells to an appropriate size vessel.  It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
5. Incubate the culture at 37°C in a suitable incubator.  A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.

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.

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

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