Jul 12, 2022
Primary cortical mixed 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 mixed culture. protocols.io https://dx.doi.org/10.17504/protocols.io.b4h6qt9e
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: January 31, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 57630
Keywords: ASAPCRN
Abstract
To obtain cortical culture, pregnant mice were anesthetized, embryos were dissected, and cortex was collected in PBS. Tissues were incubated in 0.25% trypsin–EDTA at 37°C for 15 min. Trypsinized tissue was transferred into a high-glucose DMEM/F12 medium supplemented with 10% FBS. After centrifugation (1500rpm, 5 min), the pellet was resuspended. Cells were plated onto poly(L-lysine)-coated 24-well plates at 106cells per well and cultured in NB-A.
Protocol materials
DMEM, high glucoseThermo Fisher ScientificCatalog #11965092
Fetal Bovine Serum, certified, heat inactivated, United StatesThermo FisherCatalog #10082147
Neurobasal-A MediumThermo Fisher ScientificCatalog #10888022
Fetal Bovine Serum, certified, heat inactivated, United StatesThermo FisherCatalog #10082147
For primary cortical mixed 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 5 mL ) 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 glucoseThermo Fisher ScientificCatalog #11965092
medium supplemented with 10%
Fetal Bovine Serum, certified, heat inactivated, United StatesThermo FisherCatalog #10082147
5m
Seed the cells onto poly(L-lysine)-coated 24-well plates at a cell density of 1x106 cells/well containing specialized Neurobasal-A MediumThermo Fisher ScientificCatalog #10888022 (NB-A) supplemented with 10% Fetal Bovine Serum, certified, heat inactivated, United StatesThermo FisherCatalog #10082147
After 5 days of culture, these cells are ready for further experiment