Protocol abstract
Nature Protocols 3, - 34 - 40 (2008)
Published online: 20 December 2007 | doi:10.1038/nprot.2007.477
Subject Categories: Cell and developmental biology | Cell and tissue culture | Immunological techniques | Isolation, purification and separation | Model organisms | Neuroscience
Purification and culture of nerve growth factor receptor (p75)-expressing basal forebrain cholinergic neurons
Aletta C Schnitzler1, Ignacio Lopez-Coviella1 & Jan Krzysztof Blusztajn1
Abstract
The activity of the basal forebrain cholinergic neurons (BFCNs) that innervate the cerebral cortex and hippocampus is essential for normal learning and memory. Here, we present a method to isolate and culture BFCNs from the embryonic murine septum that takes advantage of their restricted expression of the nerve growth factor receptor (p75) in conjunction with fluorescence-activated cell sorting. The septal region dissection, cell dissociation and staining process, and cell sorting parameters are described in detail. Sufficient cell yield and optimized cell culture conditions make this protocol suitable for multiple assays including immunocytochemistry, reverse transcriptase PCR, microarray profiling, acetylcholine measurements and electrophysiological assessment. The study of these neurons as a purified population will greatly advance our understanding of factors that influence their development and maintenance.
- Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Correspondence to: Jan Krzysztof Blusztajn1 e-mail: jbluszta@bu.edu
nature-products
A-Z product listing
- 0.2
m sterile filter units(Nalgene) - 0.4% (wt/vol(Sigma)
- 100 mm dishes(BD Falcon)
- 40 m nylon mesh filters(BD Falcon)
- 48-well tissue culture plates(BD Falcon)
- 50
B-27 supplement(Invitrogen)