7AC5/EYFP

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

Note: To insure the highest level of viability, pre-warm media and Trypsin/EDTA to 37ºC before adding to cells. Volumes used in this protocol are for T75 flasks. Proportionally adjust the volumes for culture vessels of other sizes. A split ratio of 1:4 to 1:7 is recommended.

Feeder Cell Preparation for Subcultures

1. Daily maintain a sufficient number of flasks that have been pre-plated with MEFs in complete medium for feeder cells.
2. One hour before subculturing the ES cells, perform a 100% medium change for the MEFs using complete growth medium for ES cells.

Dissociation and Transfer of ES Cells

1. Aspirate the medium from the flask(s) containing ES cells.
2. Wash with PBS Ca+2/Mg+2-free (ATCC® SCRR-2201).
3. Add 3.0 mL of 0.25% (w/v) Trypsin / 0.53 mM EDTA solution (ATCC 30-2101) and place in incubator. After about one minute the ES colonies will dissociate and all cells will detach from the flask.
4. Dislodge the cells by gently tapping the side of the flask then wash the cells off with 7-10 mL of fresh culture medium. Triturate cells several times with a 10 mL pipette in order to dissociate the cells into a single-cell suspension.
5. Spin the cells at 270 x g for 5 min. Aspirate the supernatant.
6. Resuspend in enough complete growth medium for ES cells to reseed new vessels at the desired split ratio (i.e. a split ratio of 1:4 to 1:7 is recommended). Perform a cell count to determine the total number of cells. ES cells should be plated at a density of 30,000 – 50,000 cells/ cm².
7. Add separate aliquots of the cell suspension to the appropriate size flask containing feeder cells and add an appropriate volume of fresh complete growth medium for ES cells to each vessel.
8. Incubate the culture at 37°C in a humidified 5% CO₂/95% air incubator. Perform a 100% medium change every day, passage cells every 1-2 days.

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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