Making Mouse Embryonic Fibroblasts (MEFs)
- Dissect E12.5-14.5 mouse embryos into a 10-cm dish with PBS. To ensure a purer fibroblastic population, remove the brain and limbs, and scoop out the internal organs (You can use some of this for genotyping.).
- Place each individual embryo in a well of a 6-well plate in PBS and transfer plate to tissue culture hood. Transfer to new sterile 6-well plate with 1 ml trypsin per well.
- Mince embryos up with a sterile razor blade (ethanol and flamed) into very small pieces, with a consistency similar to sludge. Then use 1-ml pipette to help homogenize the embryos further. Place in 37°C incubator for 10 min.
- Remove from incubator and once again use 1-ml pipette to pipet up and down to further breakup the cells. There will be some viscous material on the bottom of the plates. Immediately add 2 ml MEF media to each well.
- Split volume between 3 10-cm dishes which have been gelatinized with 0.1% gelatin and incubate at 37°C. Cells should attach within next two days. Change media once the cells have attached.
- Aspirate remaining cartilaginous chunks (very obvious when you change media).
- Grow to confluency, then freeze two 10-cm dishes as p0 and continue to grow the other plate, splitting 1:10.
- Make 0.1% gelatin solution (bovine skin type I collagen) by dissolving 0.5 g collagen into 500 ml PBS and autoclaving.
- Cover surface of tissue culture dish with gelatin and let sit at RT 1-2 hours.
- Pipet off excess solution and let dry min 15 minutes. These dishes can now be stored at 4°C indefinitely.
- 440 ml DMEM
- 50 ml FBS (heat-inactivated)
- 5 ml Pen/Strep
- 5 ml L-glutamine
- Filter sterilize
- 72 µl 14M b-ME
- 100 ml 1X PBS