Newborn mice were killed and primary cortical neurons (CNs) were isolated and cultured as previously described.23 (link) pSuper vectors containing EFhd2 short hairpin RNA (shRNA) and scrambled shRNA as well as the EFhd2-GFP vector have been described before.56 (link), 57 (link) The scrambled shRNA and the shRNA directed against EFhd2 were subcloned into pSuperNeoGFP (50) via HindIII and EcoRI restriction sites. After 7 days in culture, CNs were transfected with mRFP-ßActin together with shScramble, shEFhd2 or EFhd2-GFP using Lipofectamin 2000 (Invitrogen, Thermo Fisher Scientific, Pinneberg, Germany) and 48 h later fixed in 4% paraformaldehyde for 15 min at room temperature. Transfected CNs were analysed with a Zeiss Apotome 2 microscope (Zeiss, Jena, Germany). Data were quantified using NeuronJ (ImageJ, National Institute of Mental Health, Bethesda, MD, USA).
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Isolation and Transfection of Cortical Neurons
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Corresponding Organization :
Other organizations : Friedrich-Alexander-Universität Erlangen-Nürnberg, King's College London, Charité - Universitätsmedizin Berlin, Central Institute of Mental Health, University Hospital Heidelberg, Trinity College Dublin, University Medical Center Hamburg-Eppendorf, Universität Hamburg, Université de Montréal, Centre Hospitalier Universitaire Sainte-Justine, Medical Research Council, Commissariat à l'Énergie Atomique et aux Énergies Alternatives, CEA Paris-Saclay, University of Vermont, University of Nottingham, Inserm, Université Paris Cité, Sorbonne Paris Cité, Délégation Paris 5, Université Paris-Sud, University of Toronto, TU Dresden
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Variable analysis
independent variables
- ShRNA directed against EFhd2
- Scrambled shRNA
- EFhd2-GFP vector
- Morphology and dynamics of primary cortical neurons (CNs)
- Culture conditions of primary cortical neurons
- Transfection method (Lipofectamin 2000)
- Positive control: Scrambled shRNA
- Negative control: Not explicitly mentioned
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