Needed:

ES cell freezing medium (2x)

2x ES cell freezing medium should be made up fresh each time it is to be used, and should comprise freshly prepared 60% DMEM+, 20% FCS, and 20% DMSO (Sigma, Cat. No.D-5879).

Freezing in cryovials

The general protocol for freezing cells grown in a standard 10 cm dish at 70% confluency is given below:

1. Change media 2-3 hours before freezing the cells.

2. Freshly prepare 2x freezing media.

3. Harvest the cells in a 15 ml. tube containing DMEM+ medium after trypsinization.

4. Spin down at 1000 rpm for 5 min at room temperature.

5. Remove the supernatant then one or two drops (200 microliters) of DMEM+ medium to the tube. Shake gently but thoroughly, to disperse the cells.

6. Add an additional DMEM+ medium to a total volume of 1.5 ml and disperse the cells carefully so that they comprise a single cell suspension.

7. Add an equal volume (1.5 ml) of 2x freezing medium and mix by pipetting several times.

8. Quickly aliquot the cell suspension into three vials and immediately place them in a Styrofoam box (this will allow them to cool down gradually). Alternatively

special boxes dedicated to this task can be purchased from a number of manufacturers (for example Stratagene).

9. Place the box in a -70 0C freezer for 1-2 days, then transfer the individual cryovials into a liquid nitrogen container for long term storage.

Freezing in 96 well plates

1. Working one row at a time using a multichannel pipettor change the medium 2-3 hours prior to freezing.

2. Freshly prepare 2x cell freezing media.

3. Aspirate the medium from each well and wash the cells with PBS (approximately 200 ml).

4. Add 50 microliter trypsin to each well, then place plate in an incubator for 5-10 min.

5. Working on ice, preferably in a wide flat container, aliquot 50 microliter of DMEM+ medium into each well. Pipette the cells several times so as to get them

into a homogenous suspension.

6. Then add 100 microliter 2x cell freezing media to the wells, and again pipette to mix.

7. Finally add 80-100 microliter sterile mineral oil (Sigma, Cat. No. M-8410) to cover the cell/freezing medium mixture.

8. Wrap the plates in parafilm, place in a styrofoam box, and store in a -70 0C freezer until suchtime as the desired clones have been identified and need to be

recovered.