Isolation and Culturing of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Abstract

Source: Kim, J. Y., et al. Isolation and culture of adult neural stem cells from the mouse subcallosal zone. J. Vis. Exp. (2016)

The video demonstrates the process of isolating and culturing neural stem cells from the mouse brain's subcallosal zone. It involves dissecting tissue, enzymatic dissociation, and culturing them with differentiation factors to develop into mature neural cells.

Protocol

1. Preparation of Materials and Culture Medium

1. For the dissection and dissociation of the SCZ (Subcallosal zone), wrap the brain matrix, double-edged razor blade, and forceps with aluminum foil, and then sterilize them by autoclaving.
2. Prepare 50 ml of cold PBS (Phosphate-buffered saline) buffer to wash the whole mouse brain.
3. Set up a dissection microscope and prepare the surgical tools required for the dissection of the brain (autoclaved scissors and forceps) and the isolation of the SCZ (1 ml syringe, 30 G needle, brain matrix, and fine forceps).
4. Preparation of N2 Medium:
   1. Prepare F-12/DMEM (+L-glutamin, +sodium bi....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM/F12	Gibco	11320-033	L-glutamin, Sodium bicarbonate
Pen/Strep	Invitrogen	15140-122
N2 supplement	Gibco	17502-048
B27 supplement	Gibco	17504-044
bFGF	R&D	233-FB
EGF	Gibco	PHG0313
PBS (10X)	BIOSOLUTION	BP007a	1X dilution
HBSS	Gibco	14175-095
Dispase II	Roche	04-942-078-001
Papain	Worthington	3126
Accutase	ICT	AT-104
Fine forceps	WPI	555229F
Scissors	Storz	E3321-C
Brain matrix (1mm)	RWD	68707
Double-edged razor	DORCO	ST-300
30 gage needle	SUNGSHIM	N1300
15 ml tubes	SPL	50015
50 ml tubes	SPL	50050
35mm dish	SPL	10035	petridish
100mm dish	SPL	10090	petridish
Cover slip (18mm)	Deckglaser	111580
12 well dish	SPL	32012	non-coating
6 well dish	SPL	32006	non-coating
PLO	Sigma	P4957	0.01%
Laminin	Gibco	23017-015	10 mg/ml
Nestin	Millipore	MAB353	mouse (1:1000)
BrdU	Abcam	ab6326	Rat (1:500)
Penicillin
Streptomycin
Dissecting microscope
Centrifuge
P1000 pipette
Water bath
CO2 incubator
Trypan blue
hematocytometer