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RESEARCH DIVISION Laboratory Manual

 


 

Gamma Irradiation of PMEF's

Thawing Cells

- Remove a vial of Passage 2 Primary Mouse Embryo Fibroblasts (PMEF'S), at a concentration of 3 x 106 from liquid nitrogen and thaw quickly at 37oC.
- Add the 1ml of cell containing media to 9ml of PMEF media in a 15ml falcon tube and spin on setting one for five minutes. This procedure removes the DMSO.
- Aspirate media off the cell pellet and resuspend gently in 1ml of media using a 1ml pipette.
- Add the resuspended cells to a medium (260ml) flask containing 10ml of PMEF media.
- Incubate at 37oC for two days or until confluent. Then split 1:4 and grow until confluent.

Irradiating Cells

- Once cells are confluent tighten the lid of the flasks and take up to the gamma source in the animal house on the third floor (PMCI), if you have a number of flasks you can trypsinise all the flasks and place the cell suspensions in a falcon tube and irradiate that.
- Irradiate the flask for 41 minutes. (3000 rads required. 1 gray=1.376 min (Oct. 98), 1 gray=100 rads).
- Once irradiated, wipe flask with 70% ethanol and return to incubator until ready to trypsinise.

Trypsinising Cells

- Aspirate the PMEF media from the flask and rinse the cells with 10ml of warmed PBS, aspirating the PBS off.
- Add 2ml of warmed 0.05% trypsin/EDTA, covering the PMEF layer.
- Place the flask on the warming tray for 2 minutes, then gently tap the sides of the flask to dislodge the cells.
- Inactivate the trypsin by adding 2ml of media. Gently pipette the cells up and down to disperse any clumps and obtain a single cell suspension.
- Transfer the 4ml of cells to a 15ml falcon tube and count the number of cells.
- Spin the cells on one for 5min and resuspend at a concentration of 1 x 106 cells/ml in media containing 10% DMSO.
- Aliquot 1ml into cyrotubes and freeze in the -70oC freezer in a Mr Frosty overnight. Once frozen transfer the vials to box D2 in the -70 freezer.

 

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: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998