GAMMA IRRADIATION OF PMEF

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 warmed 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

IRRADIATING CELLS

Once cells are confluent tighten the lid of the flask and take up to the gamma source in the animal house on the third floor (PMCI)

Irradiate the flask for 38 minutes. (3000 rads required. 1 gray=1.2718 min, 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.