Changes

Fibroblasts # MEFs were prepared from tails and legs of P0 pups. Tissues from individual animals were kept separate, disaggregated with # Disect out all four limbs plus the tail. Place one small piece into PCR tube/plate for genotyping# Using a clean surgical blade and digested with 500 U/ml type II collagenase (LS004204, Worthington, US) in 5 ml RPMI media (Catchop into tiny pieces. # 1640, Invitrogen, US) supplemented with Place into 5 mL of collagenase in DMEM/PSG/10% FBS, 2 mM L-glutamine and penicillinSerum (500U/streptomycin mL) in a 15 mL falcon tube# Incubate in incubator for 12 hours at 37°C. # After incubation, cells were harvested at 200 g for 5 min and plated # Wash cells once with warm media, then resuspend in DMEM. # Plate into 2-3 100 mm dishes, in fresh RPMI medium supplemented with 10% FBS, 2 mM L-glutamine and penicillin/streptomycin. Experiments were performed on cells that were passaged 1-3 times.change media afer 1d# Split when confluent