Routine Splitting and freezing of cells

1. Grow cells to subconfluence in a flask.

2. Harvest as per normal and count.

3. Spin down 5min 1.2K in benchtop. Resuspend at 1.0 X 106/ml in 10% DMSO in media. This is prepared from frozen aliquotss of DMSO (essential to use tissue culture grade eg. DMSO Sigma D-2650, aliquoted and frozen at -70�). Filter DMSO / media prior to resuspending cells in it.

4. Add cells to Nunc cryotubes (1ml ea. Cat. 3-63401) and label with initials, experiment no. and cell name in pencil.

5. Place in a Mr Frosty (Nalgene #5100-0001) warmed to room teperature, the outer container filled with isopropanonol to the indicated line and then put container into bottom of -80� freezer.

6. Tranfer to LN2 next day and record position in cell database.