In Vitro Analysis of Mouse Mesencephalic Neural Crest Development

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

The neural crest is a unique structure in vertebrates. Neural crest cells play important roles in the formation of organs that characterize the vertebrate body plan. In this unit, we describe a primary culture method for mouse mesencephalic neural crest cells. The neural crest cells cultured by this method actively proliferate and differentiate into various cell types that originate from cranial neural crest cells, such as chondrocytes, neurons, and glia. Therefore, this primary culture method is useful for analyzing the development of mouse mesencephalic neural crest cells. Curr. Protoc. Neurosci. 56:3.23.1?3.23.8. © 2011 by John Wiley & Sons, Inc.

Keywords: mesencephalon; neural crest cells; primary culture

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Introduction
Basic Protocol 1: Primary Culture of Mouse Mesencephalic Neural Crest Cells
Support Protocol 1: Preparation of Chick Embryo Extract (CEE)
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Primary Culture of Mouse Mesencephalic Neural Crest Cells

Materials

Distilled water (DDW) sterilized by autoclave
Culture medium sterilized by filtration (see recipe )
E8.0‐8.5 mouse embryos (6 to 12 somite stages)
Hanks' balanced salt solution sterilized by filtration (HBSS; see recipe )

Collagen‐coated plastic 35‐mm tissue culture dishes, sterile
37°C humidified incubator containing 5% CO₂
60‐mm glass dishes, sterile
Stereomicroscope
Fine scissors sterilized by autoclave
Fine forceps sterilized by autoclave
Three‐well blood agglutination plate sterilized by autoclave
1‐ml disposable plastic measuring pipets, sterile
Electrically sharpened tungsten needles sterilized by flame
Disposable glass Pasteur pipets sterilized by autoclave

NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.NOTE: Steps 2 to 10, 12, 15, and 16 are performed under a stereomicroscope. All steps should be carried out on a clean bench.

Support Protocol 1: Preparation of Chick Embryo Extract (CEE)

Materials

11‐day‐old chick embryos
70% ethanol
HBSS sterilized by filtration (see recipe )
α‐modified minimum essential medium with ribonucleotides and deoxyribonucleotides (α‐MEM; Sigma, cat. no. M0644) sterilized by filtration
Ice

38°C incubator
Curved fine scissors sterilized by autoclave
Straight fine scissors sterilized by autoclave
Large forceps sterilized by autoclave
500‐, 300, and 100‐ml beakers sterilized by autoclave
100‐mm plastic petri dishes, sterile
Fine forceps sterilized by autoclave
1‐liter beaker covered with double‐layer gauze sterilized by autoclave
Large scissors sterilized by autoclave
50‐ml disposable syringe, sterile
1‐liter conical flask with a cap containing a stirring rod sterilized by autoclave
Scale
Centrifuge tubes for refrigerated centrifugation sterilized by autoclave
Centrifuge
Ultracentrifuge tubes sterilized by autoclave
Ultracentrifuge
10‐ml disposable plastic measuring pipets, sterile

NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.NOTE: Steps 3 to 11, 13, and 14 should be performed on a clean bench.

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Figures

Figure 3.23.1 Schematic diagram of mouse mesencephalic neural crest cell cultures. The lines numbered 1 to 3 in A show dissection procedures for the isolation of the mesencephalic neural fold: Separate off the cranial region of the forebrain and midbrain level by cutting at red line 1 with tungsten needles. Then, remove the forebrain region by dissecting at blue line 2. Finally, isolate the mesencephalic neural fold by dissecting at black line 3. Prepare the neural fold fragments from the isolated mesencephalic neural fold (B ). Seed the neural fold fragments in culture dishes (C ). After 24 to 48 hr, remove the epithelial components and establish the mesencephalic neural crest cell cultures (D, E ).

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Figure 3.23.2 A colony of mouse mesencephalic neural crest cells at 48 hr in culture before (A ) or after (B ) removal of epithelial components. Scale bar = 500 µm.

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Figure 3.23.3 Differentiating cells in mouse mesencephalic neural crest cell cultures. (A ) Neurons containing neurofilament 68kDa (NF68kDa) on culture day 10. (B ) Anti‐glial fibrillary acidic protein (GFAP)‐positive glial cells on culture day 4. (C ) Chondrocytes expressing collagen type II on culture day 10. Each differentiating cell was detected by immunocytochemistry using specific antibodies to NF68kDa, GFAP, or collagen type II. Scale bar = 50 µm.

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Literature Cited

Literature Cited
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In Vitro Analysis of Mouse Mesencephalic Neural Crest Development

Abstract

Table of Contents

Materials

Basic Protocol 1: Primary Culture of Mouse Mesencephalic Neural Crest Cells

Support Protocol 1: Preparation of Chick Embryo Extract (CEE)

Figures

Videos

Literature Cited