Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos

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.

Table 1: Components of 1x E2 media for zebrafish cell culture.

2. E3 Media (1x)
   1. Dissolve the components in 1 L sterile water as shown in Table 2 to make 100x stock. Dilute the stock in sterile water to make 1x E3 media.
   2. Add 0.2% methylene blue. For 20 mL of 1x E3 media, add 40 µL of methylene blue.

Table 2: Components of 100x E3 media for maintaining zebrafish embryos.

3. 80x saline stock solution
   1. Combine all components shown in Table 3. Add water to make 100 mL solution. Mix until all components are dissolved. Store the solution at 4 °C.

Table 3: Components of 80x saline solution for zebrafish cell culture media.

4. Zebrafish cell culture medium (ZFCM+)
   1. Combine all components shown in Table 4 to make 250 mL media. Adjust the pH to 7.5. Filter media using 0.22 µm filter and store at 4 °C.

Table 4: Components of zebrafish cell culture medium with serum and antibiotics.

2. Primary retinal ganglion cell culture derived from zebrafish embryos

NOTE: Perform steps 2.1 and 2.2 in a laminar flow hood.

1. Preparation of coverslips
   1. Prepare 4-6 culture plates in each experiment. Use acid-cleaned coverslips (22 x 22 mm square; 0.16-0.19 mm thickness) that are stored in 100% ethanol.
   2. Remove one coverslip from the storage container by using forceps and flame it to remove residual ethanol.
   3. Air dry the coverslip completely by placing it at an angle inside a 35-mm culture dish.
   4. Prepare Poly-D-Lysine (PDL) working solution (1x) by diluting 10x stock (5 mg/mL) in sterile water.
   5. Apply 100 µL of 0.5 mg/mL PDL to the center of each coverslip and avoid spreading of the solution to the edges.
   6. Incubate the PDL on coverslips for 20-30 min at room temperature (RT). Make sure the PDL does not dry out.
   7. Wash the PDL with 0.5 mL sterile water three times. Let the plates dry completely.
   8. Prepare laminin working solution (1x) by diluting 50x stock (1 mg/mL) in 1x PBS.
   9. Apply 100 µL of 20 µg/mL laminin in PBS to the center of each coverslip and avoid the spread of solution to the edges.
   10. Incubate the plates at 37 °C in a humidified incubator for 2-6 h. Avoid drying of the laminin solution.
2. Embryo dissection and plating RGCs
   1. Prepare and label four 35-mm tissue culture dishes and fill with 4 mL of: 70% ethanol, "E2 media 1", "E2 media 2", "E2 media 3" on the day of dissection. Keep the dishes in the fridge until dissections.
   2. When zebrafish embryos are 34 hours post fertilization (hpf), take the culture dishes coated with laminin out of the incubator and wash the coverslips three times with 0.5 mL of 1x PBS.
   3. After the final wash, add 4 mL of ZFCM(+) media to each culture dish and avoid drying the plate.
   4. Retrieve the prepared culture dishes from Step 2.2.1. Let them equilibrate to RT.
   5. Fill 4-6 PCR tubes with 15 µL of ZFCM(+) media. One tube is needed to prepare RGCs from 4 eyes to be plated onto one coverslip.
   6. Retrieve zebrafish embryos from the incubator and immerse embryos in 35 mm tissue culture dish containing 70% ethanol for 5-10 s to sterilize.
   7. Using a transfer pipette, transfer embryos to E2 Media 1 dish containing sterile E2 media to wash excess ethanol.
   8. Transfer embryos to E2 Media 2 dish and remove their chorions with sharp forceps.
   9. Transfer embryos to final E2 Media 3 dish to perform dissections.
   10. Using a pair of fine forceps, dissect out the retinas.
   11. Position and hold embryos anterior to their yolk with one of the forceps and remove the tail posterior to the yolk sac with the other forceps.
   12. Grab the neck with forceps and take off the head to expose brain and eyes to the E2 media. Avoid cutting the yolk sac.
   13. With the tip of fine forceps, gently roll the eyes off from the head, while holding the cranial tissue down with the second forceps. Keep eyes isolated from the adjacent tissue debris.
   14. Transfer four eyes to one of the previously-prepared tubes containing ZFCM(+).
   15. Gently titrate up and down with the P20 pipette and a yellow tip about 45 times to dissociate cells. Avoid any air bubbles.
   16. Transfer the ZFCM(+) with dissociated cells to the center of the coverslip. Repeat steps 10-12 for additional coverslips.
   17. Maintain cultures on benchtop at 22 °C on a polystyrene foam rack to absorb vibrations.
   18. Perform imaging 6-24 h after plating.