Isolation and Cryopreservation of Mouse Embryonic Fibroblasts

The mice used were bred at the Central Animal Facility (ZFE) of the Goethe University Medical Faculty in Frankfurt, under FELASA-certified conditions, in accordance with the ETS123 (European Convention for the Protection of Vertebrate Animals), the Council Directive of 24 November 1986 (86/609/EWG) with Annex II and the German Animal Welfare Act. Approval of the local institutional review board (Regierungspraesidium Darmstadt, project V54-19c20/15-FK/1083) was given on 27 March 2017. MEF were prepared from individual embryos at 14.5 days post-coitus of WT and Pink1^−/− mice, which were generated and bred as previously reported [20 (link)]. In brief, embryos were dissected from the uterus, extremities, and inner organs were removed and the tissue was treated with 0.05% trypsin (Gibco, Thermo Scientific, Schwerte, Germany) for 10–15 min. Cells were cultivated in Dulbecco’s Modified Eagle Medium 4.5 g/L glucose (Invitrogen, Karlsruhe, Germany) plus 15% bovine growth serum (BGS, Thermo Scientific), 1% glutamine, 1% penicillin/streptomycin (all Invitrogen,) at 37 °C and 5% CO₂ in a humidified incubator, then Pink1^−/− cells and their respective littermate WT controls were frozen in liquid nitrogen.