Mar 03, 2022
Culturing Primary Cortical Neurons
- 1University of Ottawa
Protocol Citation: Haley Geertsma 2022. Culturing Primary Cortical Neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.b5u6q6ze
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: March 03, 2022
Last Modified: March 03, 2022
Protocol Integer ID: 59006
Abstract
This protocol is used to isolate and culture primary cortical neurons from mouse embryos at E14.5-E15.5.
Day 1
Day 1
1d 3h 16m
1d 3h 16m
Coat culture dishes with 0.05mg/mL Poly-D-lysine overnight at 37oC.
1d
Day 2
Day 2
3h 14m
3h 14m
Wash culture dishes with sterile water x2 then leave to dry.
1m
Sedate pregnant mouse with 120mg/kg Euthanyl and confirm sedation prior to proceeding with the next step.
*The neurons must be plated within approximately 4 hours of sedation.
2m
Soak pregnant mouse with 70% ethanol then dissect open the abdominal cavity.
1m
Isolate uterus with embryos and place in a tube of 1X PBS.
1m
In a laminal flow hood, dissect the brains of E14.5-15.5 embryos in Hank's Balanced Salt Solution (HBSS) and isolate each brain in 700uL of HBSS.
2h
Gently pipette up/down x10 to mix cortices then add 20uL trypsin for every embryo and incubate on a rotator at 37oC for 20 minutes.
20m
Add 300uL Solution A per embryo and carefully pipette up/down x5 then centrifuge at 2500g for 5 minutes at 4oC.
Solution A: 2.75mL Neurobasal media (unsupplemented) + 150uL trypsin inhibitor + 100uL DNAse1
10m
Remove supernatant and carefully resuspend pellet in 300uL Solution B per embryo by pipetting up/down x10.
Solution B: 2.55mL Neurobasal media (unsupplemented) + 200uL trypsin inhibitor + 250uL DNAse1
5m
Centrifuge at 2500g for 5 minutes at 4oC then remove supernatant.
5m
Resuspend pellet in 1mL Neurobasal complete per embryo.
1m
Optional trypan blue cell exclusion assay: Mix 100uL trypan blue + 100uL 1X PBS + 20uL resuspended cells. Count with haemocytometer.
5m
Plate neurons on pre-coated dishes at desired confluency.
1m
Change neurobasal medium at 4 days in vitro.
5m
Fix neurons at 7 days in vitro by removing media and replacing with 4% paraformaldehyde for 10 minutes at room temperature.
10m
Wash neurons with 1X PBS x2 to remove traces of paraformaldehyde then store in 1X PBS at 4oC.
5m