Preparation of Culture Media

1. It is important that the rat and cord blood are spun as soon as possible after collection, preferably before clotting commences. The serum obtained by squeezing the fibrin clot is known as the immediately centrifuged serum.

2. Serum used as medium supplement should have a low lipid content (clear instead of cloudy). Hemolyzed serum does not support normal culture of mouse embryos.

3. The medium in the first well of the NUNC slide is used to wash the embryo once before transferring to the culture wells.

4. Growth rates decline if the embryos are overcrowded and, likewise, if they are cultured alone.

5. Best development for 48 h is achieved with embryos explanted shortly after the appearance of the primitive streak.

6. The appearance of the primitive streak provides an unequivocal indication of the anterior-posterior axis and helps with the orientation during dissection. Early gastrulation embryos are therefore used in experiments that demand knowledge of the source of cells in the anterior-posterior and medial-lateral axis. However, if only a distinction between proximal and distal epiblast is required, both pre-(without the primitive streak) and early gastrulation embryos can be used.

7. The choice of developmental stage is important in order to avoid the contamination of the cranial mesoderm by migrating neural crest cells. Previous studies have established that the first population of neural crest cells to leave the neural plate are those of the mesencephalon at the 5-6 somite stage (11-13). Therefore, in experiments involving the cranial mesoderm, only embryos having <5 somites are used so that the mesodermal explants are free of any migrating neural crest cells. In order to obtain an enriched neural crest cell population for cell fate analysis, premigratory neural crest cells are isolated from the lateral region of the neural plate of these embryos for transplantation experiments.

8. It is important that the holding pipette has a smooth surface so that the embryo is not damaged when it is held against the tip by suction.

9. If the internal diameter of the grafting pipet is large relative to the size of the embryo, it may be difficult to puncture the tissue layers with the pipet during grafting. Beveled pipets can be used in this situation. The sharp point of the beveled pipet slices the tissue layers to create a passage for the grafting pipet.

10. Do not to overheat the glass bead, since this may cause distortion and contriction of the pipet tip. It is useful to mark the side of the pipet with the bevel for later orientation.

11. The drops should be placed far apart enough to avoid mixing of contents as the pipets are moved around in the chamber.

12. When moving between the dye drop and the media drop, the holding pipet should be retracted, so that its tip does not become contaminated with the dye.

13. When drawing up the dye solution, it is best to keep the edge of the dye drop in sight, so that only the tip of the pipet is dipped into the dye drop. Pushing the pipet too far into the drop will result in excessive amount of dye adhering to the outer surface of the pipet.

14. Labeling of the embryonic tissue is best accomplished through the extraembryonic route, because it reduces the chance of accidentally labeling other germ layers. Even if dye is leaking from the pipet or coming off the surface of the pipet during the passage of the pipet, only the extraembryonic tissues will be inadvertently labeled.

15. The pipets may clog with precipitate of the dye after repeated uses. The clogged pipet can be recovered by breaking its tip by brushing against the holding pipet while in the chamber or by wiping it gently with a lint-free tissue when it has been taken out of the chamber.

16. If the cell clumps become sticky, they will follow the grafting pipet out of the embryo. Leave the pipet with the graft partly out of the tip at the transplantation site for 10-30 s to allow the graft to adhere to the surrounding tissue. Following that, a snappy retraction of the grafting pipet or tapping gently on the base plate of the manipulator may dislodge the graft from the pipet tip. Siliconizing the injection glassware also stops the cell clumps from adhering to the injection pipets.

17. A small graft is readily incorporated into the host embryo. Larger grafts may be squeezed out of the embryo as the wound closes. Pushing the tip of the grafting pipet against the graft that has been lodged in the germ layer for approx 10-30 s helps the retention of the graft.

18. Occasionally, the host embryos will collapse because of fluid leakage through the wound made to accommodate the graft. Inflating the embryonic cavity by injecting a small amount of medium to replenish the loss of fluid during transplantation may improve the development of the embryo.

19. The lids of Petri dishes are used because the rim has a lower clearance, which gives better accessibility of the angled pipets to the embryos and tissues in the dish.

20. The principle for manipulating 7.5-d embryos is the same as for 6.5-d embryos. However, manipulations are carried out in a Petri dish and this therefore requires the use of the fluovert FS inverted microscope.

21. Despite the blue-tac feet, the embryos are squashed to some extent. With 6.5- and 7.5-d embryos, it can be difficult to orient the embryos once it has been squashed. For ease of analysis, mount the embryos in the sagittal plane, and mark the direction of the anterior-posterior axis on the slide.

Was this article helpful?

0 0

Post a comment