Dissection, Plating, and Maintenance of Cortical Astrocyte Cultures Cristóvão Albuquerque, Donald J. Joseph, Papiya Choudhury, and Amy B. MacDermott 1 Department of Physiology and Cellular Biophysics and Department of Neuroscience, Columbia University, New York, NY 10032, USA INTRODUCTION Survival of central nervous system neurons in culture is usually greatly improved if the neurons are plated on top of a layer of confluent astrocytes. Under these conditions, neurons attach more firmly and develop a more readily identifiable bidimensional neuritic tree than in culture conditions where primary cells are plated on top of collagen, laminin, or other cell-free substrates. Although dissociated cells from any central nervous system region will include astrocytes and other glia, these generally do not support the survival of the primary neurons as well as preplated astrocyte feeder layers. For exam- ple, plating dorsal root ganglion (DRG)/dorsal horn (DH) cocultures directly onto collagen or laminin results in a very low number of attached DH neurons, and those have stunted neurites. Furthermore, although a higher number of DRG neurons do attach without astrocytes, this attachment is weaker, as demonstrated by the tendency of those neurons (or at least their neurites) to blow off the cover- slip when a local superfusion system is directed at them. Neonatal rat cortex provides abundant astro- cytes that can be cultured readily. The protocol presented here selects for type I astrocytes that grow in a monolayer with contact inhibition. RELATEDINFORMATION For specific protocols describing other techniques used in the preparation of neuronal cultures, see Preparation of Coverslips for Neuronal Cultures (Albuquerque et al. 2009a), Dissection, Plating, and Maintenance of Dorsal Horn Neuron Cultures (Albuquerque et al. 2009b), and Dissection, Plating, and Maintenance of Dorsal Root Ganglion Neurons for Monoculture and for Coculture with Dorsal Horn Neurons (Albuquerque et al. 2009c). © 2009 Cold Spring Harbor Laboratory Press 1039 Vol. 4, Issue 8, August 2009 1 Corresponding author (abm1@columbia.edu) Cite as: Cold Spring Harb Protoc; 2009; doi:10.1101/pdb.prot5273 www.cshprotocols.org Protocol MATERIALS CAUTIONSANDRECIPES: Please see Appendices for appropriate handling of materials marked with <!>, and recipes for reagents marked with <R>. Reagents All solutions must be sterile. <!>Cytosine β-D-arabinofuranoside (Ara-C; 1 mM, prepared in MEM; Sigma C1768) Dulbecco’s phosphate-buffered saline (D-PBS; Invitrogen 14190144) Ethanol (70% [in a squirt bottle] and 95%) <R>IMDM for astrocytes Cold Spring Harbor Laboratory Press on June 3, 2020 - Published by http://cshprotocols.cshlp.org/ Downloaded from