ZEM2S

ZEM2S cells were derived from ZEM2 in 1993 by selection for growth in a basal nutrient medium supplemented with 5 to 10% heat-inactivated fetal bovine serum.

Medium conditioned by cells from the buffalo rat liver cell line available as BRL 3A (see ATCC CRL-1442).

Both transient and stable expression of foreign genes have been demonstrated in transfected zebrafish blastula derived cell cultures.

A culture submitted to the ATCC in October 1994 was found to be contaminated with mycoplasma, and progeny were cured by a 21 day treatment with BM Cycline.

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 28°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. Resuspend content of ampule in 9 mL of complete growth medium containing 5% heat-inactivated FBS. 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 serum-free growth medium.
4. Transfer the vial contents to an appropriate size vessel. Incubate the culture at 28°C in a suitable incubator for 30 mins. If using the medium described on this product sheet, the medium formulation was devised for use in a free gas exchange with atmospheric air. A CO₂ and air mixture is detrimental to cells when using this medium for cultivation.
5. Examine to ensure attachment, and then add heat-inactivated FBS at 10% of total volume.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that 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 28°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of SERUM FREE growth medium and aspirate cells by gently pipetting.
5. To remove Trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
6. Discard supernatant and resuspend cells in fresh growth medium.
7. Add appropriate aliquots of cell suspension to new culture vessels.
8. Incubate cultures at 28°C without CO₂ for 30 minutes.
9. Examine to ensure attachment, and then add heat-inactivated FBS at 10% of total volume.
10. Incubate cultures at 28°C without CO₂

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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Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.