Modified from Attardi 10/03
Mouse Embryonic Fibroblast Protocol
First, set up timed pregnancies to collect day 12.5 or 13.5 embryos.
Materials:
DPBS: Dulbecco’s 1X PBS (Cellgro, #21-031-CV)
Trypsin: 0.05% with EDTA; without Ca and Mg (Invitrogen, #25300)
DMEM+Glutamax (Invitrogen, #10569)
FBS (not heat inactivated): Hyclone, #SH30070.03
Pen/Strep (Invitrogen, #15070)
10cm plates: (Fisherbrand, #0875712)
T75 flasks: straight neck; vented cap (BD Falcon, #353110)
T175 flasks: straight neck; vented cap (BD Falcon, #353112)
Day of Dissection:
1. Warm 0.05% trypsin-EDTA and MEF media (DMEM-Glutamax+10%FBS+1%P/S), before starting. (~30minutes)
2. Cut out embryos into a 10 cm dish (bacterial plates are fine) with DPBS. Tails can be isolated for genotyping, if desired.
3. Cut away deciduas, transfer to a new 10 cm dish with DPBS.
4. Examine embryo – dead or alive, morphology
5. Sever the head by clamping down on it with forceps or scissors. (Can save for RNA or DNA)
6. Hook/poke/scratch out the innards of the body with hooked watch-maker forceps.
7. Put body in DRY 10 cm tissue culture dish.
8. Sterilize (via dipping in alcohol and flaming) instruments between embryos.
9. Add 1 ml of warmed trypsin to the body; mince with scissors into fine pieces. (flame and sterilize in between embryos)
10. Put the plate in the 37°C incubator propped up on a pipet so that the dish is tilted and all the trypsin and embryo are on one side of the dish. Let sit 30-45 minutes .
11. Add 4mls MEF media (DMEM-Glutamax+10%FBS+1%P/S), pipet up and down about 20 times.
12. Transfer to T75’s, add 15 ml more of MEF media and let grow for 4 days to allow the cells to really pack. (Passage 1)
13. Aspirate off media, add 10 ml PBS to rinse and then aspirate that off.
14. Add 2.5-3 ml of trypsin to loosen in the incubator for 5 minutes.
15. Add 7-7.5 ml MEF media and pipet up and down, to disperse cells.
16. Collect the whole thing and transfer into 2 T175’s with a total volume of 40-50 ml per T175. Grow for 4 days. (Passage 2)
17. Trypsinize cells and freeze at 3-4X106 cells/vial in freezing media. (DMEM+10%FBS+10%DMSO)