Isolating Cranial Neural Folds From a Chick Embryo for a Neural Crest Cell Culture

Abstract

Source: Jacques-Fricke, B. T., et al. Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures. J. Vis. Exp. (2022).

This video illustrates a technique for isolating cranial neural folds from the midbrain region of chick embryos. It presents a detailed dissection process of the neural folds, their placement, and attachment on fibronectin-coated coverslips, followed by the observation of neural crest cell migration from the neural folds.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Preparation of solutions and materials Prepare Ringer's solution by mixing 123.3 mM NaCl, 1.53 mM CaCl2, 4.96 mM KCl, 0.809 mM Na2HPO4, and 0.147 mM KH2PO4 (see Table of Materials). Adjust pH to 7.4 and filter sterilize into 100 mL bottles. Stor…

Representative Results

Figure 1. Dissection of chick dorsal neural folds. Working in Ringer's P/S, spring scissors were used to excise neural folds. (A) Embryo dorsal view, anterior toward the top of the figure. Neural folds appear more opaque than the surrounding tissue. Midbrain neural folds lie posterior to the optic lobes (pink) and anterior to the cardiac crescent (yellow). (B)</strong…

Materials

AxioObserver equipped with an LSM710 confocal scan head controlled by ZEN 3.0 SR software	Zeiss		Used alpha Plan-Apochromat 100x/1.46 Oil DIC M27 objective
CaCl2	Sigma-Aldrich	C3306
Chamber dishes (glass bottom, single or divided)	MatTek; Cell Vis	P35G-1.5-14-C (MatTek)  X000NOJQGX (Cellvis) X000NOK1OJ (Cellvis)	Single chamber 35 mm or 4 chamber 35 mm
Cover glass	Carolina Biological Supply Company	633029, 633031, 633033, 633035,  633037	circles, 0.13–0.17 mm thickness, available in 12-25 mm diameter
DMEM/F12	ThermoFisher Scientific	11320033	Alternative for L15 media
Egg incubator	Sportsman	1502
FBS	Life Technologies	10437-028
Fibronectin	Fisher Scientific	CB-40008A
Filter paper	Whatman		grade 3MM chromatography
Forceps (blunt)	Fisher Scientific; Thomas Scientific	08-890 (Fisher);1141W97 (Thomas)
Forceps (fine)	Fine Science Tools	11252-20	Dumont #5
Pin holder	Fine Science Tools	26016-12	For tungsten needle (alternative for spring scissors)
KCl	Sigma-Aldrich	P3911
KH2PO4	Sigma-Aldrich	P0662
L15 media	Invitrogen	11415064
L-glutamine	Invitrogen	25030
Na2HPO4	Sigma-Aldrich	S9638
NaCl	Sigma-Aldrich	S9888
Penicillin/streptomycin	Life Technologies	15140-148	10,000 Units/mL Penicillin; 10,000 mg/mL Streptomycin
Petri Dishes	VWR (or similar)		60 mm, 100 mm
Scissors (dissection)	Fine Science Tools	14061-10
Spring Scissors	Fine Science Tools	15000-08	2.5 mm cutting edge (alternative for tungsten needle)
Sylgard	Krayden	Sylgard 184
Syringe Filters	Sigma-Aldrich	SLGVM33RS	Millex-GV Syringe Filter Unit, 0.22 µm, PVDF, 33 mm, gamma sterilized
Tissue culture dishes	Sarstedt	83-3900	35 mm culture dishes for bulk neural fold cultures
Tungsten wire	Variety of sources		0.01" diameter for tungsten needle (alternative for spring scissors)