L-WRN

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.

• 0.5 mg/mL G-418
0.5 mg/mL hygromycin B
• fetal bovine serum to a final concentration of 10%

Volumes used in this protocol are for 75 cm² flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

1. Remove and discard culture medium. Briefly rinse the cell layer with Ca⁺⁺/Mg⁺⁺ free Dulbecco's phosphate-buffered saline (D-PBS) (ATCC 30-2200) or 0.25% Trypsin – 0.53mM EDTA (ATCC 30-2101) solution to remove all traces of serum which contains trypsin inhibitor.
2. 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.

3. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
4. Add appropriate aliquots of the cell suspension to new culture vessels. Incubate cultures at 37°C.

1. Split the cells 1:10 in culture medium (without G418 and Hygromycin B to prevent carryover of drugs in the conditioned medium) and seed 25 mL cell suspension into T-150 flasks
2. Incubate the flasks for 3 or 4 days or until the cells become over-confluent and a number of cell aggregates come off.
3. Remove medium and rinse flasks with 10 mL medium and discard rinse.
4. Add 25 mL fresh medium and incubate flasks for 24 hours.
5. Remove the medium to a centrifuge tube. Add new medium to the flasks. Centrifuge the conditioned medium at 2000 x g for 5 minutes and decant supernatant into a 1 L bottle. Store the conditioned medium at 4°C. This is the first batch of medium.
6. Every 24 hours, collect 2^nd, 3^rd and 4^th conditioned medium. Centrifuge and add to same same bottle.
7. After the 4^th collection, add an equal volume of fresh medium to the bottle (final concentration: 50%), mix well and aliquot media into 50 mL centrifuge tubes and store at -20°C.
8. Collect 5^th to 8^th and 9^th to 12^th conditioned media, if desired.

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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