Isolation and Culture of Mouse Fibroblasts

Primary mouse embryonic fibroblasts (MEFs) were isolated from wild-type (WT) and AOX-transgenic (AOX^Rosa26) [7 (link)] embryos (E13.5–15.5) in the C57BL/6 background and immortalized (generating iMEFs) as described elsewhere [35 (link), 36 (link)]. MEFs, iMEFs, and human lung carcinoma (A549) cells (86012804, Sigma-Aldrich/Merck Life Science) were grown in Dulbecco Modified Eagle’s Medium (DMEM) supplemented with 4.5 g/l glucose (BE12-614F, Lonza/BioNordika), 10% fetal bovine serum (FBS, 10270106, Gibco/ThermoFisher Scientific), 100 U/ml penicillin plus 100 μg/ml streptomycin (P0781, Sigma-Aldrich/Merck Life Science), and 2 mM L-glutamine (BE17-605E, Lonza/BioNordika). Mouse pulmonary artery smooth muscle cells (PASMCs) were isolated from precapillary pulmonary arterial vessels of WT C57Bl/6 mice as previously described [37 (link)]. PASMCs were cultured in Smooth Muscle Cell Growth Medium (C-22062, PromoCell) supplemented with 10% FCS (F7524, Sigma-Aldrich/Merck Life Science) and 0.002% Normocin (ant-nr-1, InvivoGen).

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Giordano L., Aneja M.K., Sommer N., Alebrahimdehkordi N., Seraji A., Weissmann N., Rudolph C., Plank C., Jacobs H.T, & Szibor M. (2021). Alternative oxidase encoded by sequence-optimized and chemically-modified RNA transfected into mammalian cells is catalytically active. Gene Therapy, 29(12), 655-664.

Publication 2021

P0781 C-22062 F7524 Normocin (ant-nr-1,

A549 cells Cultured medium Eagle Embryonic Fibroblasts Glucose Glutamine Human Lung carcinoma Mice Penicillin Pulmonary artery Smooth muscle cell Streptomycin Transgenic

Corresponding Organization :

Other organizations : Tampere University, Tampere University of Applied Sciences, Tampere University Hospital, Ethris (Germany), Cardio-Pulmonary Institute, University of Giessen, Universities of Giessen and Marburg Lung Center, German Center for Lung Research, Helsinki Institute of Physics, University of Helsinki

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Variable analysis

independent variables

Genotype (wild-type vs. AOX-transgenic)

dependent variables

Not explicitly mentioned

control variables

Cell type (primary mouse embryonic fibroblasts (MEFs), immortalized MEFs (iMEFs), human lung carcinoma (A549) cells, mouse pulmonary artery smooth muscle cells (PASMCs))
Culture medium (Dulbecco Modified Eagle's Medium (DMEM) for MEFs, iMEFs, and A549 cells; Smooth Muscle Cell Growth Medium for PASMCs)
Supplements (4.5 g/l glucose, 10% fetal bovine serum, 100 U/ml penicillin plus 100 μg/ml streptomycin, 2 mM L-glutamine for MEFs, iMEFs, and A549 cells; 10% FCS, 0.002% Normocin for PASMCs)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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