Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos

Abstract

Source: Terzi, A., et al., ROS Live Cell Imaging During Neuronal Development. J. Vis. Exp. (2021)

The video demonstrates a method for culturing primary retinal ganglion cells from zebrafish embryos. Early-stage embryos undergo dissection to isolate the eyes, which are subsequently mechanically dissociated into a suspension of retinal ganglion cells. This cell suspension is cultured on coverslips coated with adhesive proteins to facilitate axon development.

Protocol

1. Preparation of solutions

1. E2 media (1x)
   1. Prepare 100x E2A (500 mL), 500x E2B (100 mL) and 500x E2C (100 mL) solutions by combining all components shown in Table 1. Autoclave E2A, E2B and E2C solutions. Store at 4 °C.
   2. For 1x E2 media: Combine 5 mL of 100x E2A, 1 mL of 500x E2B, and 1 mL of 500x E2C. Bring the volume to 500 mL with sterile water. Adjust the pH to 7.0-7.5.
   3. Prepare 50 mL aliquots of 1x E2 media and store at -20 °C for long-term storage. However, precipitations can occur upon thawing. Make sure precipitation is fully dissolved before using the stock solution.
   <....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35-mm culture dish	Sarstedt	83-3900
Calcium Chloride Dihydrate	Fisher Scientific	C79-500
Cover glass	Corning	2850-22
Disposable Petri Dishes (100 x 15 mm)	VWR	25384-094
Fetal Bovine Serum	ThermoFisher Scientific	26140087
Glucose	Sigma Aldich	G7528
HEPES	Sigma Aldich	H4034
Laminin	ThermoFisher Scientific	23017-015
Leibovitz's L-15 Medium without phenol red	Gibco/Fisher Scientific	21-083-027
PBS	Hyclone/Fisher Scientific	SH3025601
Penicillin/streptomycin	ThermoFisher Scientific	15140122
Poly-D-Lysine (PDL)	Sigma Aldich	P7280
Sodium Pyruvate	Sigma Aldich	P5280
Steritop 0.22 μm filter	Millipore	S2GPT05RE