Generation and Differentiation of Primary Neurospheres from Zebrafish Neural Stem Cells

Abstract

Source: Lopez-Ramirez, M. A. et al., Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres. J. Vis. Exp. (2016)

This video demonstrates the generation and differentiation of primary neurospheres. Neural stem cells from zebrafish, in the presence of growth factors, proliferate into free-floating neurospheres with self-renewal capacity, which later differentiate into glial cells and neurons.

Protocol

1. Preparations Prepare 10 ml of dissection medium, and add 200 µl of 100x penicillin-streptomycin into 9.8 ml of Dulbecco's Modified Eagle Medium F12 (DMEM/F12). Prepare L-Cysteine solution: to 10 ml of water for tissue culture, and add 120 mg of L-cysteine. Store the L-cysteine solution at 4 °C for up to 2 weeks or in aliquots at -20 °C for up to 1 year. Prepare DNase I solution: to 1 ml of water for tissue culture, add 10 mg of DNase I. Store the DNas…

Representative Results

Figure 1: Differentiation of Zebrafish Brain-derived Neurospheres. (A-C, E) Phase contrast images of whole brain-derived neurospheres cultured in the Z-differentiation medium during 1 (A, DiVd1), 3 (B, DiVd3) and 4 (C and D, DiVd4) days. (E) D…

Materials

DPBS 1X	Life Technologies	14190-144
DMEM/F12 1X	Life Technologies	11330-032
L-Cysteine hydrochloride monohydrate	Sigma	C6852-25g
B-27	Life Technologies	17504-044
N-2	Life Technologies	17502-048	N-2 supplement (100x) liquid
HEPES	Life Technologies	15630	1M
D-(+)-Glucose 45%	Sigma	G8769
Penicillin-streptomycin	Life Technologies	15140-122
Fetal Bovine Serum	Life Technologies	16000044
Human FGF-basic	Peprotech	100-18B
Human EGF	Peprotech	AF-100-15
Insulin	Sigma	I5500-50 mg
DNAse	Sigma	DN25-10mg
Papain	Worthington Biochemical Corporation	LS003126
Matrigel	Becton Dickinson	356234
PFA	TCI	P0018
PBS	AmericanBio	AB11072-04000
Tricaine MS-222	Sigma	A5040	Stock solution of 4 mg/ml.
Trycold gel	Sigma	TGP8	gel pack
Trypan blue stain	Life Technologies	15250061