Isolation of Mouse Embryonic Fibroblasts (MEFs)

From Zolov et al (under review)

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

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