Plating cells from liquid nitrogen storage

Aim:

To retrieve 1mL cryo-tube cell aliquots from liquid nitrogen storage and plate in a T25 flask.

Perform all work in the sterile laminar flow cell cabinet.

Steps

1. Before removing cells from liquid nitrogen, pre-fill 10 or 15mL tubes with 6mL of cell media. These tubes must fit your centrifuge. Pre-fill one tube for every cell aliquot to be removed from liquid nitrogen.

2. Remove cells from liquid nitrogen & transport on ice. (Dry ice is best for longer distances as dry ice is colder).

Note: As cells may be frozen in media with a cryo-protecting chemical (e.g. DMSO) that may be toxic to cells, it is important to move / work very quickly once cells start to thaw.

3. Once thawing begins quickly transfer cells to the tubes pre-filled with 6mL of media.

4. Centrifuge at 1000 rpm for 5 minutes & pour off media. (This step removes DMSO etc). Gently resuspend cells in 7mL media and transfer each tube of cells to a new T25 flask. Incubate at 37°C for 24 hours then change media to remove any dead cells.

For quick growth you may transfer 2- 3 tubes to a single T25 flask.