Dejellying embryos (Sive et al. 2000)

From XenWiki
Jump to navigation Jump to search

Information adapted from:

Early Development of Xenopus Laevis: A Laboratory Manual.[1]

2000; (First ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

Sive, Hazel (Author), Grainger, Robert M (Author), and Harland, Richard M (Author).

Dejellying embryos

1. Dejelly embryos by removing buffer and swirling gently in 1x modified Marc's Ringer (MMR), or water with 2% (w/v) cysteine at pH 8.0

2. Gently swirl the eggs for 2-4 minutes undid the jelly membranes are visible in the solution and the eggs have started to pack. Dejellying is usually complete in 4 minutes, but is frog-dependent and must be triturated for each animal. It is safest to monitor each dish of embryos during the process.

Note: Prolonged exposure to cysteine solution will damage embryos. In addition, cysteine solutions become less potent during the day and each batch of embryos must be monitored during dejellying.

3. When the eggs begin to pack together closely and fragments of jelly can be seen floating in the buffer, promptly decant the cysteine and rinse the fertilized eggs 10 times in 0.1x modified Barth's saline (MBS), over a period of approximately 10 minutes. It is essential to rinse embryos thoroughly in a clean beaker to remove all traces of cysteine.

Note: The vitelline membrane thickens during the first 30 minutes postfertilization and thus, if possible, it ie best not to dejelly until after this stage; the thickened membrane offers some protection to the fragile embryo. Embryos are particularly sensitive to the dejellying process during gastrulation and neurula stages and must be closely monitored to avoid excessive treatment.

4. After dejellying, place embryos in a clean dish. Keep embryos at low density (100 per 80-mm petri dish) in 0.1x MBS and remove dead embryos promptly.