(Source: D. Frost and D. Sepich from Zebrafish Book 5th Edition)
1. Rinse embryos in sterile, calcium-free Ringer's solution.
2. Transfer about 10-100 embryos in a single drop of calcium-free Ringer's solution to the center of a tissue culture plate.
3. Drop a sterile coverslip onto the embryos and smash them.
4. Add more calcium-free Ringer's solution, remove the coverslip and suspend the cells by swirling the plate.
5. Transfer the suspension to a sterile centrifuge tube and spin at 300 x g for 7 minutes.
6. Remove most of the supernatant, triturate the pellet through a sterile narrow-bore Pasteur pipette to resuspend the cells and then add several ml of growth medium.
7. Centrifuge, 300 x g, 7 minutes.
8. Remove supernatant and resuspend in enough growth medium to make a final concentration of 15 fish per ml.
9. Plate the cells and incubate at 28.5°C.