Co‐Culture of Neurons and Microglia

Pamela J. Roqué1, Lucio G. Costa2

1 Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, 2 Department of Neuroscience, University of Parma, Parma
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 11.24
DOI:  10.1002/cptx.32
Online Posting Date:  November, 2017
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Microglia, the resident immune cells of the brain, have been implicated in numerous neurodegenerative and neurodevelopmental diseases. Activation of microglia by a variety of stimuli induces the release of factors, including pro‐ and anti‐inflammatory cytokines and reactive oxygen species, that contribute to modulating neuro‐inflammation and oxidative stress, two crucial processes linked to disorders of the central nervous system. The in vitro techniques described here will provide a set of protocols for the isolation and plating of primary cerebellar granule neurons, primary cortical microglia from a mixed glia culture, and methods for co‐culturing both cell types. These methods allow the study of how microglia and the factors they release in this shared environment mediate the effects of toxicants on neuronal function and survival. The protocols presented here allow for flexibility in experimental design, the study of numerous toxicological endpoints, and the opportunity to explore neuroprotective strategies. © 2017 by John Wiley & Sons, Inc.

Keywords: co‐culture; glia; microglia; neurons; neuro‐inflammation; oxidative stress

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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation and Culture of Mouse Primary Cerebellar Granule Neurons
  • Basic Protocol 2: Preparation of Cortical Mixed Glial Cultures
  • Basic Protocol 3: Isolate Primary Microglia from Mixed Glial Cultures
  • Basic Protocol 4: Primary Neuron and Microglia Co‐Cultures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Isolation and Culture of Mouse Primary Cerebellar Granule Neurons

  • Mice, post‐natal day (PND) 6 to 8
  • 40 µg/ml poly‐D‐lysine working solution (see recipe)
  • Hank's balanced salt solution (HBSS; see recipe)
  • Papain wosrking solution, 1 ml per 2 pups (see recipe)
  • 4 mg/ml DNase (see recipe)
  • NB medium (see recipe)
  • Hank's balanced salt solution, calcium and magnesium free (HBSS‐CMF; see recipe)
  • NB minus antioxidants (NB–AO)
  • 24‐well sterile cell culture plates
  • 12‐mm, round glass coverslips (optional)
  • 15‐ and 50‐ml conical tubes
  • 50‐ml tubes (2) containing 70% ethanol, two‐thirds full
  • 5‐ and 10‐ml serological pipettes
  • Sterile 35‐mm dish for tissue collection
  • 10‐ml syringe with 0.22 syringe filter for sterilization of papain
  • Sterile filter paper, round, 100‐mm
  • Sterile 100‐mm dishes (3) for pup and tissue collection, with filter paper
  • Ice in a small container to hold 35‐mm dish during tissue collection
  • Sterile surgical tools
  • 100‐µm filters for 50‐ml tube

Basic Protocol 2: Preparation of Cortical Mixed Glial Cultures

  Additional Materials (also see protocol 1)
  • Mice, post‐natal day 3 (PND3) pups
  • Mixed glia and microglia medium (see recipe)
  • 0.25% trypsin in PBS (see recipe), 1 ml per pup cortex
  • Phosphate‐buffered saline (PBS; see appendix 2A)
  • T175 flasks
  • Laminar‐flow cell culture hood

Basic Protocol 3: Isolate Primary Microglia from Mixed Glial Cultures

  Additional Materials (also see Basic Protocols protocol 11 and protocol 22)
  • Established mixed glia cultures, days 12 to 15 (see protocol 2)
  • 24‐well inserts (3‐µm pores)
  • Tweezers to move inserts

Basic Protocol 4: Primary Neuron and Microglia Co‐Cultures

  Additional Materials (also see Basic Protocols protocol 11 and protocol 22)
  • Primary CGNs, day 10 (see protocol 1)
  • Primary microglia in inserts, day 2 (see protocol 3)
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Literature Cited

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