Sep 09, 2022

Public workspacePreparation of primary rat cortical neuron and astrocyte co-culture

DOI
dx.doi.org/10.17504/protocols.io.n92ldzq1xv5b/v1
  • 1Queen Square UCL Institute of Neurology;
  • 2The Francis Crick Institute
Minee L Choi
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DOI: dx.doi.org/10.17504/protocols.io.n92ldzq1xv5b/v1
Protocol Citation: mineechoi 2022. Preparation of primary rat cortical neuron and astrocyte co-culture . protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldzq1xv5b/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: May 25, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 63211
Keywords: ASAPCRN
Abstract
This protocol describes how to prepare primary rat cortical neuron and astrocyte co-culture.
Materials
  • Dissecting buffer: Hanks' Balanced Salt solution (HBSS) supplemented with Concentration10 millimolar (mM) HEPES and Concentration20 % volume Fetal bovine serum (FBS)

  • Washing buffer: HBSS supplemented with 10mM HEPES

  • Disgesting buffer: Concentration0.5 Mass / % volume EDTA-trypsin supplemented with DNAse

  • Neurobasal completed medium: Neurobasal A medium supplemented with B27, Concentration2 millimolar (mM) Glutamax, Pen/Strep

1-3 days postpartum Sprague Dawley rats (University College London breeding colony) are used.
Experimental procedures are performed according to the United Kingdom Animal (Scientific Procedures) Act of 1986.
Rat cortices are placed in an ice-cold Dissecting buffer (described in Materials).
Wash five times with Washing buffer (described in Materials).
Tissues are digested with a Disgesting buffer (described in Materials) for Duration00:15:00

15m
Digested tissues are neutralized with a dissecting buffer.
Washing twice with a Washing buffer,
Dissociate with a Washing buffer supplemented with DNAse.
Dissociated pellets are collected in Neurobasal completed medium.
Approximately 600,000 cells are plated onThikness25 mm Poly-D-Lysin (PDL) coated coverslips and 200,000 cells for 8-well ibidi chambers (PDL coated).

The cultures are maintained at Temperature37 °C (Concentration5 % volume CO2), and the media are changed every 4-5 days.
Cells can be used at 12-16 days.