Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos
Published: July 31, 2024
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 E2 media (1x) 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. 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. Prepare 50 mL aliquots of 1x E2 media and store at -20 °C for …
Declarações
The authors have nothing to disclose.
Materials
| 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 |
Citar este artigo
Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos. J. Vis. Exp. (Pending Publication), e22371, doi: (2024).