C57BL/6

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.

Complete Medium for Feeder Cells

Feeder cells may be grown in medium containing fewer growth factors than those required by the ES cells. Feeder cells are available from ATCC. Consult the product sheet provided for the feeder cells you wish to use for medium requirements. 

Feeder cells should be initiated 24-48 hours prior to inoculating with embryonic stem (ES) cells.

Feeder Cells

ATCC recommends culturing ES-C57BL/6 on mouse embryonic fibroblasts (MEFs) that have been mitotically arrested by either irradiation or treatment with Mitomycin-C. ES-C57BL/6 cells have been cultured on mitotically arrested MEF (CF-1) (ATCC^® SCRC-1040™).

1. At least one day before plating the ES cells, prepare the desired combination of flasks with feeder cells to accommodate an initial ES cell seeding density of 30,000 cells/cm² to 50,000 cells/cm².
2.  Refer to the batch specific information provided on the last page of the product information sheet for the total number of viable cells recovered from this lot of ATCC^® SCRC-1002™.
3.  Plate mitotically arrested mouse embryonic fibroblasts (MEFs) as a feeder layer at approximately 55,000 feeder cells/cm² in complete medium for feeder cells.
4.  Refer to the product sheet for mitotically arrested MEF for detailed handling instructions.
5.  Feeder cells should be used within one week of plating. It is best to use feeder cells within 24 to 48 hours of initiation.

Embryonic Stem (ES) Cells

1. 30 Minutes Prior to Handling Cells – Pre-warm complete growth medium for ES cells at 37°C for at least 30 minutes before adding to cells.
2. One Hour Prior to Thawing the ES Cells – Perform a 100% medium change for the MEFs using complete growth medium for ES cells.
3. Thaw the vial of ES cells 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 90 seconds).
4. Remove the vial from the water bath before the contents are completely 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.
5. Transfer the vial’s contents plus 5 mL of complete growth medium for ES cells to a 15 mL centrifuge tube. Use an additional 1 mL of media to rinse the vial and transfer the liquid to the 15 mL tube. Add 4 ml of complete growth medium for ES cells to bring the total volume to 10 mL.
6. Spin the cells at 270 x g for 5 min. Aspirate the supernatant and resuspend the pellet in 2 mL of complete growth medium for ES cells.
7. Add the 2 mL of cell suspension to the appropriate size flask containing feeder cells and fresh complete growth medium for ES cells (see batch specific information). ES cells should be plated at a density of 30,000 – 50,000 cells/cm².
8. Incubate the culture at 37°C in a humidified 5% CO₂/95% air incubator. 

Routine Handling

Perform a 100% medium change every day. Passage the cells every 1 to 2 days. If the colonies are close to or touching each other the culture is overgrown. Overgrowth will result in differentiation.

Make sure that you have prepared a sufficient number of flasks pre-plated with MEF feeder layers to support frequent passage of the ES cells.

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.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

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