Jul 13, 2022
Primary cortical neuronal culture
- Xiqun Chen1,
- Qing Ye2
- 1Department of Neurology Massachusetts General Hospital Harvard Medical School Charlestown MA 02129 USA, Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network Chevy Chase MD 20815 USA;
- 2Department of Neurology Massachusetts General Hospital Harvard Medical School Charlestown MA 02129 USA, Department of Neurology Longhua Hospital Shanghai University of Traditional Chinese Medicine Shanghai 200032 China
Protocol Citation: Xiqun Chen, Qing Ye 2022. Primary cortical neuronal culture. protocols.io https://dx.doi.org/10.17504/protocols.io.b4t7qwrn
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: February 08, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 57951
Keywords: ASAPCRN
Abstract
Primary cortical neurons were prepared from C57BL/6J mice embryonic day 17. The dissected cortical tissue was digested, triturated, and centrifuged. Cells were plated onto poly (L-lysine)-coated 24-well plates at 106 cells per well and cultured in NB-A with 2% B27 (Invitrogen, USA). After 24 h in culture, 5µg/ml cytarabine was added to inhibit the growth of glial cells in the medium and then changed to the original medium 48 h later. Neurons were cultured for 5 days and ready for experiments.
Materials
DMEM/F12 DMEM, high glucoseThermo Fisher ScientificCatalog #11965092
FBS Fetal Bovine Serum, certified, heat inactivated, United StatesThermo FisherCatalog #10082147
Poly L Lysine
Neurobasal-A medium Neurobasal-A MediumThermo Fisher ScientificCatalog #10888022
B27 B-27™ Supplement (50X), serum freeGibco - Thermo FisherCatalog #17504044
Cytarabine
Protocol materials
Fetal Bovine Serum, certified, heat inactivated, United StatesThermo FisherCatalog #10082147
Neurobasal-A MediumThermo Fisher ScientificCatalog #10888022
B-27™ Supplement (50X), serum freeGibco - Thermo Fisher ScientificCatalog #17504044
DMEM, high glucoseThermo Fisher ScientificCatalog #11965092
Poly-L-Lysine
Neurobasal™-A MediumThermo FisherCatalog #10888022
B-27™ Supplement (50X), serum freeGibco - Thermo Fisher ScientificCatalog #17504044
DMEM, high glucoseThermo Fisher ScientificCatalog #11965092
Fetal Bovine Serum, certified, heat inactivated, United StatesThermo FisherCatalog #10082147
For Primary cortical neuronal culture - Use C57BL/6J mice at embryonic day 17
Anesthetized pregnant mice (1% sodium pentobarbital, 80mg/kg), dissect their embryos and collect the cortex.
(Separate and remove the soft membrane and blood vessels, rinse the cerebral cortex in PBS, and use the ophthalmic scissor to cut pieces of the cortex)
Collect the cortices in PBS in a 50 ml tube on ice
(The 50 ml tube contains 30 ml of PBS) On ice
Transfer the cortices to 15 ml tubes containing 1.5 ml trypsin–EDTA (0.25%) and incubate it at 37 °C for 00:15:00 Dissociate the cortices by triturating with a 10 mL serological pipette 10 – 15 times
15m
Centrifuge the dissociated cortices (1500 rpm , 00:05:00 ) and resuspend the pellet in 10ml DMEM, high glucoseGibco, ThermoFisherCatalog #11965092 medium supplemented with 10% Fetal Bovine Serum, certified, heat inactivated, United StatesGibco, ThermoFisherCatalog #10082147
5m
Triturate the cell suspension 10 times with a 1ml pipette
Coat 24 well plate with Poly-L-LysineGibco, ThermoFisher (PLL)
Place the cover glass at the bottom of the 24-well plate and add 400ul/well of PLL coating ( 0.01%) for 24h and incubate at 37 °C
Wash 3 times with PBS after 24:00:00
1d
Seed the cells onto PLL coated 24-well plates at a cell density of 106 cells/well containing specialized Neurobasal™-A MediumGibco, ThermoFisherCatalog #10888022 supplemented with 2% B-27™ Supplement (50X), serum freeGibco, ThermoFisherCatalog #17504044
Incubate for 24:00:00 at 37 °C
1d
Post 24 h, add 2µg/well of cytarabine (Stock - 5µg/ml) to the culture to inhibit the glial cell growth.
48:00:00 later remove the medium completely
Add 400µl/well of NB-A supplemented with 2% B27
2d
Transduction with BRAF (Optional)
After 5 days of culture, these cells are ready for further experiment
After 5 days, the neurons were transduced with BRAFV600E, or BRAFWT, or vector lentivirus with 8 μg/ml polybrene (Sigma–Aldrich, USA) for 24 h.
The cells were cultured in NB-A for 120 h and used for subsequent experiments.