Home Neuroscience Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device
Neuroscience JoVE (Open Access) Citable · DOI

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

DOI: 10.3791/305-v
What you'll learn
  • Isolate and prepare primary cortical neurons from E18 rat embryos
  • Dissociate cortical tissue using enzymatic digestion and mechanical trituration
  • Load neurons into microfluidic devices for axonal compartmentalization
  • Perform axotomy to study axonal regeneration in vitro
Protocol

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

Difficulty
advanced
Total time
~3–4 hours per preparation (embryo isolation through device loading)
Model organism
Rat (Sprague Dawley or Lewis), E18 fetal cortex
Biosafety
BSL-1

Steps

1
Review protocol and biological context

Understand the rationale for E18 cortical neuron isolation and microfluidic compartmentalization for axonal regeneration studies.

▶ 00:03
2
Prepare tools, reagents, and workspace

Sterilize dissection instruments, prepare enzymatic digestion solutions, and set up the microfluidic device and culture plates.

▶ 00:47
3
Enzymatically digest E18 fetal rat cortex

Incubate isolated cortical tissue in papain solution to dissociate cells from the tissue matrix.

▶ 01:46
4
Mechanically dissociate cortical tissue by trituration

Pipette the digested tissue through progressively smaller-bore pipette tips to separate individual neurons.

▶ 04:14
5
Load neurons into microfluidic compartmentalization device

Plate dissociated neurons into the microfluidic device at appropriate density to establish axonal growth into separate channels.

▶ 07:26
6
Image compartmentalized neurons under fluorescence microscopy

Visualize axon and soma compartments in the microfluidic device using phase contrast or fluorescent markers.

▶ 08:56
7
Perform axotomy for axonal regeneration assay

Mechanically sever axons within the microfluidic device using a focused laser or micropipette to initiate regeneration studies.

▶ 09:37
💬 Comments coming soon