Mar 03, 2025
Primary Neuron Cultures
- Emily Tabaie1
- 1University of California, Riverside
Protocol Citation: Emily Tabaie 2025. Primary Neuron Cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4wpyovo5/v1
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, 2025
Last Modified: March 03, 2025
Protocol Integer ID: 123717
Abstract
This protocol is for the isolation and preparation of primary cortical neuron cultures from the embryos of C57BL/6 mice.
Primary Neuron Cultures
Primary Neuron Cultures
Set up a breeder pair 18 days prior to harvest
When the female is close to giving birth coat a 6 well plate days before the dissection occurs and make the media
Prior to coating, sterilize coverslips by autoclaving
Using sterile forceps, transfer one pre-sterilized glass coverslip per well of a 6 well plate
Dispense 1-2 mL of Poly-L-Lysine (PLL) solution into each well containing a coverslip
Incubate for a minimum of 2 hours at 37°C or overnight at 4°C
Wash each well with 1 mL Tissue Grade Water two times
Add 2 mL of complete neurobasal media to each well of a 6 well plate
Prepare Sacrifice pregnant female by isoflurane/cervical dislocation
Surgically open peritoneum and remove embryonic sacks
Prepare 1950 μL of Enzyme Mix 1 for up to 400 mg of tissue (5 brains)
Pre-heat the mixture at 37°C for 10-15 min prior to use
Carefully open each sack and separate the embryo in a petri dish filled with cold HBSS without Ca2+ or Mg2+
Remove the head and the skullcap to excise the brain and get rid of any red blood cells
Separate the forebrain from the cerebellum and place the forebrain into a 15 mL conical tube containing 14 mL of HBSS (without Ca2+ or Mg2+)
Centrifuge at 300 x g for 2 min at room temperature and aspirate the supernatant
Add 1950 μL of enzyme mix 1
Incubate in a closed tube for 15 min at 37°C under slow, continuous rocking/rotation
Prepare 30 μL of enzyme mix 2 per eppendorf tube and add to the 15 mL conical and invert the tubes gently to mix
Dissociate tissue mechanically by passing through an 18 gauge needle 10 times
Incubate in a closed tube for 10 min at 37°C under slow, continuous rocking/rotation
Dissociate tissue again by passing through an 20 gauge needle 10 times
Incubate in a closed tube for 10 min at 37°C under slow, continuous rocking/rotation
Apply the cell suspension to a 40 m strainer placed on a 50 mL tube
Wash the filter with 10mL HBSS (with Ca2+ or Mg2+) (Sigma 55037C)
Centrifuge cell suspension at 300 x g for 10 min at room temperature and aspirate supernatant
Resuspend pellet in 2 mL of warm Neurobasal Media and count cells
Plate 500,000 cells per well in a 6 well plate
Change ONLY 50% of the media every three days with Neurobasal media without glutamic acid