BNL CL.2

The line was selected to grow in medium containing ornithine and phenylalanine in place of arginine and tyrosine.

Tested and found negative for ectromelia virus (mousepox).

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 by rapid agitation in 37°C water bath. Thawing should be rapid (within 40-60 seconds). As soon as the ice is melted, remove the ampule from the water bath and immerse in 70% ethanol at room temperature. All of the operations from this point on should be carried out under strict aseptic conditions.   
2. Transfer the cell suspension and dilute it with the recommended culture medium in a culture flask (see specific batch information above for dilution ratio); incubate at 37°C with 10% CO₂ in air atmosphere. Since it is important to avoid excessive alkalinity of the medium during recovery of the cells, it is suggested that the culture medium be placed into the culture flask, tube, etc. and the pH be adjusted, as necessary, prior to the addition of the ampule contents. Note that the bicarbonate content of the culture medium will determine whether an atmosphere containing CO₂ will be required.   
3. It is not necessary to remove the freezing additive. However, if desired, the culture medium may be changed to remove the protective freezing additive (dimethylsulfoxide) 24 hours after thawing. If it is desired that the freezing additive be removed immediately, or that a more concentrated cell suspension be obtained, centrifuge the above diluted suspension at approximately 125 x g for 10 minutes, discard the fluid and resuspend the cells with growth medium at the dilution ratio given in the specific batch information.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3. 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 15 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.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.

Subcultivation Ratio: 1:2 to 1:6
Medium Renewal: Every 2 to 3 days

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a Manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.

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.

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

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