For freezing, determine how many 35 mm dishes worth of cells you have. Each vial contains the equivalent of one 35 mm dish (i.e. 2 vials per 60 mm or 6 vials per 100 mm). Allow cells to grow highly confluent and feed four hours before freezing.

-To freeze, trypsinize using fresh trypsin just as if passaging (see above) and put into an equal volume of ES cell culture medium.

-Spin down as usual and break up pellets promptly

-Resuspend in 1.5 ml of 1 x CPM per vial. Gently resuspend cells and put into labeled vials. Put vials on ice until all the vials are ready to freeze.

{1 x CPM is plain IMDM with 10% serum and 10% DMSO. Add serum to the IMDM first and swirl. Add the DMSO, swirl, then 0.2 µ filter. 1 x CPM can be frozen in 7 ml aliquots and thawed just before use (keep on ice). Always freeze the aliquots in a frost-free freezer first, to insure that they all freeze completely. After that they can be stored in the Tissue Culture -20 wrapped in foil to protect from light.}

-When all the vials are ready to freeze, put them in the isopropanol freezing box (kept under tissue culture sink when not in use--note number of uses and replace with fresh isopropanol after 5 uses). Promptly put the box in the -80. After 24 hours the vials can be put in a convenient freezer box and kept at -80 for at least another 24 hours before transferring to liquid nitrogen. They can stay in -80 longer than that (up to several months in fact) but need at least 48 hours to acclimate to extreme cold.

-The isopropanol box is normally stored under the tissue culture sink and takes several hours to thaw out. If you really need it, it can be thawed in the water bath or in a tub of water. It should be at room temperature before use. It sometimes happens that you have more than the 18 vials that fit into the isopropanol box (for instance, when making feeders). While I wouldn't do this with your most precious stocks of ES cells, you can put the extra vials inside a pair of 15 ml falcon tube racks. Wrap the racks together with tape and put in the -80 as usual. I've never found anything wrong with this. It is in fact what many labs do routinely to achieve a gradual temperature drop (approximately 1 degree per minute) to minus 80 degrees.

-When ready to place vials in the liquid nitrogen dewar, keep vials on dry ice or inside the isopropanol box still frozen at -80. Since I top off the dewar on Fridays, I often wait to put vials in until that day, then put in fresh liquid nitrogen.