Sep 06, 2022
Primary neuronal cultures
- Miguel Da Silva Padilha1,
- Irina Dudanova1,
- F. Ulrich Hartl1,
- Itika Saha2,
- Mark S. Hipp3,4
- 1Molecular Neurodegeneration Group, Max Planck Institute of Neurobiology, 82152 Martinsried, Germany;
- 2Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;
- 3Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Antonius Deusinglaan, 1, 9713 AV Groningen, The Netherlands;
- 4School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, 26129 Oldenburg, Germany
Protocol Citation: Miguel Da Silva Padilha, Irina Dudanova, F. Ulrich Hartl, Itika Saha, Mark S. Hipp 2022. Primary neuronal cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1ojwklr2/v1
Manuscript citation:
Itika Saha, Patricia Yuste-Checa, Miguel Da Silva Padilha, Qiang Guo, Roman Körner, Hauke Holthusen, Victoria A. Trinkaus, Irina Dudanova, Rubén Fernández-Busnadiego, Wolfgang Baumeister, David W. Sanders, Saurabh Gautam, Marc I. Diamond, F. Ulrich Hartl, Mark S. Hipp bioRxiv 2022.02.18.481043; doi: https://doi.org/10.1101/2022.02.18.481043
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 05, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 69593
Keywords: ASAPCRN
Abstract
This protocol describes the preparation of primary neuronal cultures from E15.5 CD-1 wild type mouse embryos. Experiments involving animal models must be performed in accordance with relevant institutional guidelines and regulations.
Sacrifice pregnant female mice by cervical dislocation.
Remove the uterus from the abdominal cavity and place into a 10 cm sterile Petri dish on ice containing dissection medium, consisting of Hanks’ balanced salt solution (HBSS) supplemented with 0.01 M HEPES, 0.01 M MgSO4 and 1% penicillin/streptomycin.
Isolate each embryo, decapitate the heads, remove the brains from the skull and immerse in ice-cold dissection medium.
Dissect cortical hemispheres, and remove meninges under a dissection microscope.
Collect the cortices in a 15 mL sterile tube and digest with 0.25% trypsin containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 15 μL 0.1% DNAse I for 20 min at 37 °C.
Stop digestion by removing the supernatant and washing the tissue twice with Neurobasal medium (Invitrogen) containing 5% Fetal Bovine Serum.
Resuspend the tissue in 2 mL Neurobasal medium and triturate to achieve a single cell suspension.
Spin cells at 130 x g, remove the supernatant, and resuspend the cell pellet in Neurobasal medium with 2% B-27 supplement (Invitrogen), 1% L-glutamine (Invitrogen) and 1% penicillin/streptomycin (Invitrogen).
Plate cells at desired density in dishes or coverslips coated with 1 mg/mL poly-D-lysine (Sigma) and 1 µg/mL laminin (Thermo Fisher Scientific).