ES-D3 [D3]

The cells spontaneously differentiate into embryonic structures in the absence of a feeder layer or conditioned medium. They can be injected back into blastocysts and contribute to the germline.

Undifferentiated cells can be genetically modified by gene targeting techniques.

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.

NOTE:  Cells must be maintained on feeder layers of irradiated (12,000 rads) or mitomycin C treated (0.01 mg/ml for 90 minutes) primary mouse embryonic fibroblasts or STO cells. (see ATCC CRL-1503, STO or ATCC 56-X, irradiated STO cells). Plate feeder layers at 2 x 10⁶/T25 or 6 x 10⁶/T75 one or two days before plating ES-D3 cells.

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. Transfer the vial contents to a centrifuge tube containing  9.0 mL complete culture medium. and spin at approximately 125 x g for 5 to 7 minutes.
4. Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio). and dispense into a 25 cm² or a 75 cm2 culture flask with feeder layer.
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.

Volumes are given for a 75 cm² flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.

Note: The cells can be maintained in the undifferentiated state by frequent subculture on feeder layers of mitomycin C (MITC) treated (0.01 mg/mL for 90 minutes) primary mouse embryonic fibroblasts or STO cells (see ATCC CRL-1503, STO or ATCC 56-X.2, MITC-STO cells).

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 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 fresh medium, aspirate and dispense onto fresh feeder layer cultures.
6. Incubate cultures at 37°C.

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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