Millipore Technical Publications |
 | Protocol - MCPROTO034 | |
Protocol: Isolation of Primary Mouse Embryonic Fibroblasts (PMEFs)insert subtitle here | ||
| Lit No: | MCPROTO034 | |
| Year: | 2007 | |
Requirements
- 13.5 to 14.5 ED pregnant mouse (inbred mice that are resistant to selection agent)
- Sterile surgical instruments (scissors, forceps)
- Phosphate buffer saline (PBS)
- 10cm tissue culture petri dishes
- Glass Pasteur pipette
- Trypsin/EDTA
- DMEM + 10% FBS +1% pen/strep+ 1% L-glutamine
- 154cm2 tissue culture standard flasks
- Laminar flow hood.
- Sacrifice the pregnant mouse by CO2 asphyxiation.
- Lie the mouse on its back and swab with 70% ethanol. Using scissors make a cut across the belly and cut away the skin to expose the gut. With sterile forceps and scissors, dissect out the uterus and place it into a petri dish with sterile PBS.
- Isolate the embryos from the uterus, and release the embryos from the embryonic sacs. Transfer embryos to a second petri dish with sterile PBS.
- Remove the embryo heads and limbs and scoop out the liver, intestines and heart with a pair of forceps. Transfer the embryo carcass into a sterile 15mL tube with a sufficient volume of Trypsin/EDTA to cover the carcasses. Using scissors finely mince the tissue.
- Incubate the tissue for 15 minutes at 37°C, then pipette the tissue a few times through a glass Pasteur pipette to dissociate the tissue. Allow the large pieces of cellular debris to settle. Remove the supernatant into a fresh tube and add 50 mL of fibroblast media. Transfer to a 154cm2 flask. Plate out about 5 embryos/flask. Incubate at 37°C with 5% CO2.
- PMEFs should attach and begin to divide in 1-3 days. After 2 days change the medium which should be very acidic (indicated by the media turning yellow in color).
- When the flask are confluent, usually in 3-4 days, the cultures are ready for freezing. Freeze cells in 10% DMSO at 2x106 cells/vial (labeled P0).