Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Summary

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Abstract

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Introduction

Neurons are complex cells that carry information essential for a variety of functions including sensation, vision, motor movement, learning, and memory. Unique from other cell types, neurons extend arm-like processes, called axons, to form essential neural highways for communication. During development specialized compartments located at the tips of growing axons, called growth cones, navigate through a concert of extracellular cues to lead the axon to its appropriate destination. The intricate molecular mechanisms that underlie growth cone navigation are not fully understood. To better understand these mechanisms, investigators have used cell culture models to study ....

Protocol

1. Coverslip Preparation: Acid Wash and Bake

1. At least 2 days prior to dissection (step 2), begin the following steps to prepare coverslips. Perform the acid wash step to remove oils and thoroughly clean coverslips.
2. Load coverslips in porcelain holder that vertically positions coverslips and prevents them from touching each other. Submerge holder and coverslips into glass histology container filled with 2 M hydrochloric acid (HCl). Alternatively, if porcelain holder is not available, spread ~20 coverslips horizontally to cover the bottom of 100 mm glass Petri dish and submerge coverslips in 2 M HCl.
3. Place glass vessel (with coversli....

Results

The protocol described here enables investigators to culture an enriched population of dissociated embryonic sensory neurons with very few (e.g., <5%) non-neuronal cells^7,8,10,16. Numerous DRGs can be obtained from the lumbosacral, thoracic and cervical regions. Depending upon the needs of the investigator, DRGs from these distinct anatomical regions can be easily isolated. For example, Figure 1 shows images of the chick embryo through various stages of dissection with the lumbar D.......

Discussion

Here we present detailed protocols for isolating and culturing dissociated sensory neurons from a chick embryo. This procedure generates an enriched population of robustly growing neurons in vitro^7,8,10,16. Numerous cellular and molecular techniques can be applied to these cultured neurons, including immunocytochemistry, which is described here. This protocol was recently used to quantitatively assess the intensity of immunolabeled activated integrins in sensory growth cones^8,16. In these p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sterile small culture dishes (35 mm)	Corning	430165
Sterile large culture dishes (100 mm)	Falcon	353003
Sterile large Petri dishes (100 mm)	VWR	89000-302
Glass Petri dish (100 mm)	VWR	D108962
Fine forceps	Fine Science Tools	11251-10
Standard forceps	Fine Science Tools	1100-12
Coverslips (22 x 22 mm)	Fisher	12 518 105K	0.13-0.17 mm thickness is optimal for microscopy
Porcelin coverslip holder	Thomas scientific	8542E40
Fetal Bovine serum	Gibco	17502-048	use 50 ml aliquots per 500 ml of Ham's F12 media
HCl	VWR	VW3204-1	use at 2 M, can be used twice before discarding
NaCl	Sigma	S9888	use 5 M NaCl solution for laminin-1 stock solution
Laminin-1	Invitrogen	23017-015	Add 72 µl of sterile 5 M NaCl and aliquot into 50 µl
15 ml Conical tube	cell treat	229411
PBS CMF 10x	Gibco	14200	used at 1x, dilute with sterile Millipore filtered water
PBS 10x	Gibco	14080	used at 1x, dilute with sterile Millipore filtered water
Ham's F12	Lonza	12-615F
Fetal Bovine Serum Albumin (BSA)	Gibco	10438	use in F12HS20 at 10%
HEPES	Sigma	H3375	make sterile 1 M solution in distilled water, dilute in F12 media to obtain final concentration of 10 mM
Penicillin/streptomyocin	Sigma	P0781	use 5 ml in 500 ml of F12 media
NT3	Millipore	GF031	Add 1 ml of sterile Millipore water, aliquot under sterile conditions, store at -20 °C, use at final concentration of 10 ng/ml in F12H media, keep in -20 °C non-defrosting freezer
NGF	RnD systems	256-GF	Add 1 ml of sterile 1x PBS with 0.1% BSA, aliquot under sterile conditions, store at -20 °C, used at final concentration of 10 ng/ml in F12H media
L-Glutamine	Sigma	G7513	aliquot under sterile conditions, store at -20 °C, use at final concentration of 200 mM in F12H media
Trypsin	Sigma	T4049	aliquot under sterile conditions, store at -20 °C
Bovine Serum Albumin (BSA)	Gibco	15260
Other items needed: general dissection instruments, including glass Pasteur pipettes, fertile white leghorn chicken eggs, check egg incubator (humidified, 37 °C), cell incubation chamber (humidified, 37 °C), laminar flow hood, binocular stereovision dissecting scope
Immunocytochemistry Reagents Table
Sucrose	Sigma	S9378	used in fixative solution (4% paraformaldehye, 30% sucrose, 2x PBS)
Triton-X 100	Sigma	T-8787
Paraformaldehyde	Sigma	P6148	used in fixative solution (4% paraformaldehye, 30% sucrose, 2x PBS)
Fluoromount G	Southern Biotech	0100-01
Normal goat serum	Life technologies	PCN500
Microscope slides	VWR	16004-430
Primary Antibody Table
Antibody against NCAM	Millipore	AB5032	polyclonal
Antibody against activated β1 ingegrin	Millipore	MAB19294	monoclonal
Secondary Antibody Table
Goat anti-mouse IgG Alexa 488	Life technologies	A11001
Goat anti-rabbit IgG Alexa 548	Life technologies	A11036