MEF Media
50ml serum
5ml Pen/Strep
5ml 200mM Glutamine

Mouse Embryonic Fibroblast Collection from Embryos
NOTE: perform procedure in cell culture hood and use aseptic technique collection at dpc13.5 (or 12.5)
1. place freshly harvested embryos in 10cm cell culture dish
2. cover embryos in PBS and remove placental and other maternal tissues
3. cut away top of head (eye and above) and eviscerate removing all innards + blood vessels
4. save head for DNA isolation for genotyping (or yolk sac or liver)
5. place embryo body in separate 6cm plate
6. mince embryo with razor blade
7. add 1ml trypsin and transfer suspension to 12well plate
8. place 12well plate in 378C incubator for 15-20min
9. quench typsin activity by adding some MEF media (DME w/ 10% FCS+P/S) in well
10. pipette 10-20x to break up tissues
11. transfer cellular suspension to T75 flask or 10cm plate and add 6-15ml MEF media
12. allow cells to grow to confluency (3-4days)
13. aspirate off medium and rinse with 10ml PBS
14. add 3ml trypsin and incubate in 378C incubator 5-10min
15. quench by adding 7ml MEF media
16. pipette 15x to resuspend cells
17. transfer suspension to 2 T175 flasks and add 50ml MEF media to each flask
18. allow cells to grow to confluency (3-4 days)
19. harvest cells via trypsin and freeze down cells @ 3x106 cells/vial

3T3 Senescence Assay (MEFs)
1. aspirate media and wash dish(es) with 5ml PBS @RT
2. add 500ul trypsin (diluted with PBS) to each dish
3. incubate @378C for 5min
4. agitate plates to observe proper effects of typsin and quench with 2ml MEF media
5. add 2.5ml more MEF media with a 5ml pipette
6. using the 5ml pipette break cell clumps to single cells by pipetting 5x
7. pellet cells: 1000rpm, 3min, RT
8. resuspend pellet in 3ml MEF media using a 5ml pipette
9. count cells using a hemacytometer
10.replate 3*105 cells in duplicate on 60mm plates in 3ml total volume

Low Density Plating (MEFs)
1.plate 1000 cells into a p100
2.feed cells as normal every 2-3days

Proliferation Assay (MEFs)
1.plate a total of 3x10^5 cells onto a 12 well dish, on day 0 (2.4e4 cells/well)
2.feed cells as normal every 2-3 days, after day 5 feed every other day
3.count a single (or up to three) well of cells on days 1, 3, 5, 9, 13, 17 etc.

Focus Frequency Formation Assay (MEFs)
NOTE: mutant cells are mixed with WT or feeder (nonproliferative) cells
1.plate 3e5 WT/feeder cells per p60 and add 3e4 mutant cells in duplicate serial dilutions of important experimentals to plate 3e3 and 3e2 also in duplicate
3.incubate cells as normal feeding every 2-3days and eventually stain with Giemsa stain

Soft Agar Assay
1. make 0.5% LMP-agar by diluting 3% stock into prewarmed 37¾C DME-HEPES
2. add 5ml 0.5% LMP-agar to each p60
3. place p60(s) in non-shaking refridgerator 30min to solidify
4. place p60(s) on benchtop for 30min to allow plates to equilibrate to RT
5. trypsinize MEFs to be plated and count
6. resuspend pellet(s) in 15% FCS serum MEF media @1x10^5 cells/ml
7. in duplicate add 5x10^4 cells per p60 suspended in 0.34% LMP-agar in 15% FCS
8. place on benchtop @RT at least 30min to allow for plates to solidify
9. place dishes in 37¾C/5%CO2 incubator

Tumor formation in nude mice
Trypsinize 10 millions cells in one ml of PBS – inject 100 µl under the skin of nude mice – wait 1-2 weeks for tumor formation.

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Last modified: October 10, 2005