Primary Neuron Culture from Embryonic Rats

Prepare Dissociation and Neutralization media fresh and cool 15mL HBSS (+Mg/Ca) on ice.

Sacrifice time-pregnant rat (E18) by CO₂ asphyxiation followed by secondary method (thoracotomy or cervical dislocation).

Note: All procedures are performed in compliance with AAALAC guidelines and are approved by the Biogen Institutional Animal Care and Use Committee.

Wash lower abdomen with 70% EtOH and make incision to reveal embryos.

One at a time, remove embryos from amniotic sac and decapitate. Cut below brain on rostral or caudal side and gently peel off skull. Remove brain and transfer to ice-cold HBSS. Repeat until desired amount of tissue is collected (~5x10⁶ cells/brain). Decapitate any unused embryos.

Optional: If there are significant delays between collection and dissection, you may use ice-cold Hibernate EB Complete Media (BrainBits HEB500) to extend viability. Delays between collection and dissection will impact cell viability.

Transfer brains to 10cm petri dish with ~5ml ice-cold HBSS.

Separate hemispheres, remove meninges and olfactory bulb, and dissect out desired tissue. Immediately transfer tissue to ice-cold HBSS and store on ice until all tissue is dissected.

Decant or aspirate HBSS and add 10mL warm Dissociation Media (up to 10 brains/10mL). Rock tissue at 37^oC from 30 minutes.

Allow tissue to settle and decant or aspirate Dissociation Media. Add 10mL warm Neutralization Media.

Remove all but 2mL of Neutralization Media, and gently triturate using P1000 pipette 10 times (avoid bubble formation). Let sit 2 minutes at RT.

Transfer supernatant (containing cell suspension) to new 15mL falcon tube, and add 2mL of Serum Media to the remaining settled tissue.

Repeat step 9, and then add the second cell suspension to the previously collected cells.

Spin down cell suspension at 30xg for 5 minutes, and then resuspend in 10mL of Serum Media.

Assess cell count and viability, and plate as needed on poly-D-lysine coated cultureware with NB Media.

Perform 50% media change using NB media every 4-7 days depending on seeding density.

Optional: Add FUDR at DIV4 to inhibit mitotic cell growth.