Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from a Mouse Embryo

Abstract

Fujiki, R., et al. Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Isl^mn:GFP transgenic mice. J. Vis. Exp. (2019)

The video demonstrates the isolation of oculomotor, trochlear, and spinal motor neurons from a transgenic mouse embryo. The embryo expresses motor neurons labeled with a green fluorescent protein (GFP) for easy visualization and identification. The motor neurons are surgically isolated under a fluorescence microscope and maintained in a culture medium.

Protocol

1. Ventral midbrain and Spinal Cord Dissection NOTE: Perform all of the following steps except for steps 1.1.1−1.1.3 and 1.1.5−1.1.6 under a fluorescence dissection stereomicroscope. Total dissection time per experiment is typically 3−5 h, depending on the proficiency at the dissection technique and the number of motor neurons required for each experiment. Ventral midbrain dissection Euthanize a pregnant mouse app…

Representative Results

Table 1: Appropriate volumes of papain, albumin-ovomucoid, and final suspension used in dissociation steps. The appropriate volumes of papain and albumin-ovomucoid to be used with various numbers of ventral midbrain and ventral spinal cord tissues were modified from the manufacturer's instructions after several rounds of optimization. Because tissues are subject to stress during dissociation and sorting, a pooled collection of more than 10 ventral midbrains and more than three ventral spinal cor…

Materials

B27 Supplement (50X), serum free	Thermo Fisher Scientific	17504-044
BD FACSAria llu SORP Flow Cytometer	BD Bioscience	–	This has 4 laser system equipped with 405, 488, 594, and 640 nm lasers.
BD Falcon 70μm Nylon Cell Strainers	CORNING	352350	For filtering the dissociating cells before FACS.
BD Falcon Round Bottom Test Tubes With Snap Cap	CORNING	352054
BDNF Human	ProSpec-Tany TechnoGene, Ltd.	CYT-207
Cell Culture microplate, 96 well, PS, F-bottom (Chimney Well)	Greiner Bio-One International	655090	We tried multiple 96-well dishes and this was the best one for culture and analyses after ICC
CNTF Human	ProSpec-Tany TechnoGene, Ltd.	CYT-272
4′,6-diamidino-2-phenylinodole (DAPI)	Thermo Fisher Scientific	D1306
Dimethyl sulfoxide	Sigma-Aldrich	D2650	DMSO
Dumont #5 Forceps Inox Tip Size .05 x .01 mm Biologie Tips	Roboz Surgical Instrument	RS-5015
Forskolin	Thermo Fisher Scientific	BP25205
GDNF Human	ProSpec-Tany TechnoGene, Ltd.	CYT-305
GlutaMAX supplement	Thermo Fisher Scientific	35050-061
Hanks' Balanced Salt Solution (HBSS)	Thermo Fisher Scientific	14175-095
Hibernate E	BrainBits	HE
Hibernate E low fluorescence	BrainBits	HELF	Fluorescence which hinders observation of embryo's GFP expressions should be low.
Horse serum, heat inactivated, New Zealand origin	Thermo Fisher Scientific	26050-070
IBMX	Tocris Cookson	2845	Isobutylmethylxanthine
Laminin	Thermo Fisher Scientific	23017-015
Leibovitz's L15 medium	Thermo Fisher Scientific	11415064
2-Mercaptoethanol	Sigma-Aldrich	M6250
Micro Dissecting Scissors	Roboz Surgical Instrument	RS-5913
Micro Knife 4.75" 1.7 x 27 mm blade	Roboz Surgical Instrument	RS-6272
Moria Mini Perforated Spoon	Fine Science Tools	10370-19
Olympus 1.7ml Microtubes, Clear	Genesee Scientific	22-281	These are the tubes that we described "1.7 mL microcentrifuge tubes" in the context.
Papain Dissociation System	Worthington Biochemical Corp	LK003150	Papain solution and alubumin- ovomucoid inhibitor solution are prepared from this kit.
Penicillin-streptomycin (10,000 U/ ml)	Thermo Fisher Scientific	15140-122
Phosphate buffered saline (PBS)	Thermo Fisher Scientific	10010-023
Poly D-lysin (PDL)	MilliporeSigma	A-003-E
SMZ18 and SMZ1500 zoom stereomicroscopes with DS-Ri1 camera	Nikon	–	Dissection was performed and images of dissected embryos and tissues are captured under these fluorescence microscopes.
Sylgard 170 Black Silicone Encapsulant – A+B 0.9 Kg kit	Dow Corning	1696157	We make dissection dishes using this kit.
TC treated Dishes, 100 x 20 mm	Genesee Scientific	25-202	We make dissection dishes using this dish.
Thum Dressing Forceps 4.5" Serrated 2.2 mm Tip Width	Roboz Surgical Instrument	RS-8100
Zeiss LSM 700 series laser scanning confocal microscope and Zen Software	Carl Zeiss	–	Confocal image of the embryo was captured with these equipments