In 96Well Plates

1. Working one row at a time using a multichannel pipetter, change the medium 23 h prior to freezing.

2. Freshly prepare 2X cell-freezing media.

3. Aspirate the medium from each well, and wash the cells with PBS (approx 200 pL).

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

5. Working on ice, preferably in a wide, flat container, aliquot 50 pL of DMEM+ into each well. Pipet the cells several times in order to get them into a homogenous suspension.

6. Then add 100 pL 2X cell freezing media to the wells, and again pipet to mix.

7. Finally add 80-100 pL 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°C freezer until such time as the desired clones have been identified and need to be recovered (see Note 17).

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