Isolation of Mouse Embryonic Fibroblasts (MEFs)

From Zolov et al (under review)

Materials

• Collagenase, type II (LS004204, Worthington, US)
• Clean surgical blades
• DMEM/PSG/10% FBS

Generation of Mouse Primary Fibroblast Cells

1. MEFs were prepared from tails and legs of P0 pups.
2. Disect out all four limbs plus the tail. Place one small piece into PCR tube/plate for genotyping
3. Using a clean surgical blade, chop into tiny pieces.
4. Place into 5 mL of collagenase in DMEM/PSG/10% Serum (500U/mL) in a 15 mL falcon tube
5. Incubate in incubator for 12 hours at 37°C.
6. After incubation, cells were harvested at 200 g for 5 min
7. Wash cells once with warm media, then resuspend in DMEM.
8. Plate into 2-3 100 mm dishes and change media afer 1d
9. Split when confluent