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. |
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