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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 -70C). 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 -80C freezer.
  6. Tranfer to LN2 next day and record position in cell database.
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail:
David Bowtell PMCI October 1998